Substrate analogs for MurG, methods of making same and assays using same

ABSTRACT

General methods for monitoring the activity of MurG, a GlcNAc transferase involved in bacterial cell wall biosynthesis, is disclosed. More particularly, the synthesis of simplified substrate analogs of Lipid I (the natural substrate for MurG), which function as acceptors for UDP-GlcNAc in an enzymatic reaction catalyzed by MurG, is described. Assays using the substrate analogs of the invention are further disclosed, which are useful for identifying a variety of other substrates, including inhibitors of MurG activity, for facilitating mechanistic and/or structural studies of the enzyme and for other uses. High throughput assays are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.09/241,862, filed Feb. 2, 1999, now U.S. Pat. No. 6,413,732, whichclaimed benefit under 35 U.S.C. §119(e) to U.S. Provisional ApplicationSerial No. 60/073,376, filed Feb. 2, 1998. All applications are herebyincorporated by reference in their entireties.

1. FIELD OF THE INVENTION

The present invention relates to substrate analogs of aUDP-GlcNAc:muramyl pentapeptide pyrophosphoryl,N-acetylglucosaminyltransferase (GlcNAc transferase, MurG, or itshomologs), an enzyme involved in bacterial cell wall biosynthesis. Thesubstrate analogs of the invention are usefull as functional substitutesof Lipid I, the membrane bound, natural substrate of MurG. Inparticular, the substrate analogs of the present invention can be usedadvantageously in an assay for the enzymatic activity catalyzed by MurG,in methods for identifying other substrate analogs of MurG, as well asinhibitors of enzymatic activity or cell wall biosynthesis (i.e.,potential antibacterial drugs), and in the isolation/purification ofMurG, including studies of its active protein/peptide fragments.

2. BACKGROUND OF THE INVENTION

2.1. Bacterial Enzymology

The emergence of resistance to existing antibiotics has rejuvenatedinterest in bacterial enzymology. It is hoped that detailed mechanisticand structural information about bacterial enzymes involved in criticalbiosynthetic pathways could lead to the development of new antibacterialagents. Because interference with peptidoglycan biosynthesis is a provenstrategy for treating bacterial infections, all of the enzymes involvedin peptidoglycan biosynthesis are potential targets for the developmentof new antibiotics. While some detailed structural and mechanisticinformation on some of the early enzymes in the pathway is nowavailable, most of the downstream enzymes have proven very difficult tostudy.

There are two main reasons for this difficulty: First, the downstreamenzymes are membrane-associated, making them intrinsically hard tohandle; secondly, discrete substrates for most of the downstream enzymesare either not available or not readily so. In some cases monomericsubstrates are difficult to obtain in large quantities from naturalsources. In other cases substrates, which may be available in largequantities from natural sources, are intractable polymeric substances.In the absence of readily available discrete substrates, it has beenimpossible to develop enzyme assays that can be used to measure theactivity of the downstream enzymes reliably and under a well-defined setof reaction conditions. This unfulfilled need has thwarted attempts topurify many of the downstream enzymes in an active form suitable forstructural characterization, much less permitted attempts to obtaindetailed mechanistic information on such enzymes.

Some of the best antibiotics function by interfering with thebiosynthesis of the peptidoglycan polymer that surrounds bacterialcells. With the emergence of bacterial pathogens that are resistant tocommon antibiotics it has become imperative to learn more about theenzymes involved in peptidoglycan biosynthesis. Although remarkableprogress has been made in characterizing some of the early enzymes inthe biosynthetic pathway (See, e.g., (a) Fan, C.; Moews, P. C.; Walsh,C. T.; Knox, J. R. Science 1994, 266, 439; (b) Benson, T. E.; Filman, D.J.; Walsh, C. T.; Hogle, J. M. Nat. Struct. Biol. 1995, 2, 644; (c) Jin,H. Y.; Emanuele, J. J.; Fairman, R.; Robertson, J. G.; Hail, M. E.; Ho,H. T.; Falk, P.; Villafranca, J. J. Biochemistry 1996, 35, 1423; (d)Skarzynski, T.; Mistry, A.; Wonacott, A.; Hutchinson, S. E.; Kelly, V.A.; Duncan, K. Structure 1996, 4, 1465; (e) Schonbrunn, E.; Sack, S.;Eschenburg, S.; Perakis, A.; Krekel, F.; Amrhein, N.; Mandelkow, E.Structure 1996, 4, 1065. (f) Benson, T. E.; Walsh, C. T.; Hogle, J. M.Biochemistry 1997, 36, 806.), the downstream enzymes have provenexceedingly difficult to study. Part of the difficulty stems from thefact that such downstream enzymes are membrane-associated (See, e.g.,(a) Gittins, J. R.; Phoenix, D. A.; Pratt, J. M. FEMS Microbiol. Rev.1994, 13, 1; (b) Bupp, K.; van Heijenoort, J. 1993, 175, 1841.), makingthem intrinsically hard to handle, and partly because substrates formany of the enzymes are not readily available. (See, e.g., (a) Pless, D.D.; Neuhaus, F. C. J. Biol. Chem. 1973, 248, 1568; (b) van Heijenoort,Y.; Gomez, M.; Derrien, M.; Ayala, J.; van Heijenoort, J. J. Bacteriol.1992, 174, 3549.) These problems have impeded the development ofactivity assays suitable for detailed mechanistic investigations of thedownstream enzymes. For a fluorescent assay to monitor MraY activity,see: Brandish, P. E.; Burnham, M. K.; Lonsdale, J. T.; Southgate, R.;Inukai, M.; Bugg, T. D. H. J. Biol. Chem. 1996, 271, 7609.

2.2. MurG

One such downstream enzyme is MurG, which is involved in peptidoglycanbiosynthesis. MurG catalyzes the last intracellular step in thebiosynthetic pathway of peptidoglycan biosynthesis, i.e., the transferof UDP-N-acetylglucosamine (UDP-GlcNAc) to the lipid-linkedN-acetylmuramylpentapeptide substrate, Lipid I. (See, Scheme 1, below.)

Although the murG gene is first identified in E. coli in 1980 and issequenced independently by two groups in the early 1990's, very littleis known about the MurG enzyme. There are no mammalian homologs, and nodirect assays for MurG activity have been developed, in part because thelipid-linked substrate (Lipid I, Scheme 1) is extremely difficult toisolate. This lipid-linked substrate is present only in minutequantities in bacterial cells. Although it is possible to increase thequantities of lipid-linked substrate by using bacterial cells engineeredto overexpress enzymes involved in the synthesis of the lipid-linkedsubstrate, isolation remains very difficult. Moreover, the isolatedsubstrate is hard to handle.

Consequently, MurG activity is currently assessed using crude membranepreparations by monitoring the incorporation of radiolabel fromradiolabeled UDP-GlcNAc donor group into lipid-linked acceptorcomponents in the membrane. To increase the signal, the membranes areoften prepared from bacterial cultures that overexpress MraY and/orMurG. MraY is the enzyme that catalyzes the reaction that attaches theMraY substrate, UDP-N-acetyl muramic acid pentapeptide, to a lipidphosphate moiety to provide Lipid I, which is the substrate for MurG.Typically, the membrane preparations are supplemented with exogenousUDP-N-acetyl muramic acid pentapeptide for conversion to Lipid I. ThisMraY substrate can be readily isolated in large quantities frombacterial cultures. Although this “coupled” enzyme assay is manageablefor screening of potential inhibitors of the MurG enzyme, it is notsuitable for detailed mechanistic investigations, and it cannot be usedto follow MurG activity during purification.

More specifically, MurG is a cytoplasmic membrane-associated enzymewhich catalyzes the transfer of UDP-N-acetylglucosamine (UDP-GlcNAc) tothe C4 hydroxyl of an undecaprenyl pyrophosphate N-acetylmuramylpentapeptide substrate (Lipid I), resulting in the assembly of thedisaccharide-pentapeptide building block (Lipid II, Scheme 1), which isincorporated into polymeric peptidoglycan. See, e.g., (a) Bugg, T. D.H.; Walsh, C. T. Nat. Prod. Rep. 1992, 199; (b) Mengin-Lecreulx, D.;Flouret, B.; van Heijenoort, J. J. Bacteriol. 1982, 151, 1109. Asalready mentioned, the muramyl pentapeptide substrate is unique tobacteria Hence, the MurG enzyme is a potential target for the discoveryor design of specific MurG inhibitors.

Despite decades of effort spent characterizing MurG activity, there isvirtually no structural or mechanistic information on the enzyme. See,e.g., (a) Anderson, J. S.; Matsuhashi, M.; Haskin, M. A.; Strominger, J.L. Proc. Natl. Acad. Sci. USA 1965, 53, 881; (b) Anderson, J. S.;Matsuhashi, M.; Haskin, M. A.; Strominger, J. L. J. Biol. Chem. 1967,242, 180; (c) Taku, A.; Fan, D. P. J. Biol. Chem. 1976, 251, 6154; (d)Mengin-Lecreulx, D.; Texier, L.; van Heijenoort, J. Nucl. Acid. Res.1990, 18, 2810; (e) Ikeda, M.; Wachi, M.; Jung, H. K.; Ishino, F.;Matsuhashi, M. Nucl. Acid Res. 1990, 18, 4014; (f) Mengin-Lecreulx, D.;Texier, L.; Rousseau, M.; van Heijenoort, J. J. Bacteriol1991, 173,4652; (g) Miyao, A.; Yoshimura, A.; Sato, T.; Yamamoto, T.; Theeragool,T.; Kobayashi, Y. Gene, 1992, 118, 147; (h) Ikeda, M.; Wachi, M.;Matshuhashi, M. J. Gen. Appl. Microbiol., 1992, 38, 53. Difficultiesisolating Lipid I have prevented the development of a simple, directassay for MurG activity. Consequently, it has not been possible topurify MurG in a quantifiably active form or to determine the minimalfunctional length; nor has it been possible to carry out any detailedmechanistic studies, or to determine the substrate requirements.

Therefore, there exists a need for a direct enzyme assay that can beused both for effective screening of enzyme inhibitors and for thepurification, characterization and identification of MurG, its variousmutants and active fragments thereof.

3. SUMMARY OF THE INVENTION

Substrate analogs for MurG enzyme, a GlcNAc transferase, are disclosed.For the first time, a substrate analog of Lipid I, as shown above inScheme 1, (i) having a structure that is accepted by at least wild typeMurG enzyme such that a labeled coupling product is produced by theGlcNAc transferase activity of the enzyme in the presence of thesubstrate analog and labeled UDP-GlcNAc, and (ii) having structuralfeatures that facilitate the separation of labeled UDP-GlcNAc from thelabeled coupling product.

In particular, a substance is described herein, which comprises thechemical moiety of the formula:

in which “R” is an acyl group comprising 2 or more carbon atoms, “R₁” isa substituted or unsubstituted alkyl group comprising 1 or more carbonatoms, “R₂” is a hydrogen or a substituted or unsubstituted alkyl groupcomprising 1 or more carbon atoms, “A” is a substituted or unsubstitutedamino acid residue or a peptide comprising 2 or more substituted orunsubstituted amino acid residues, “R₃” is a substituted orunsubstituted alkyl group comprising 5 or more carbon atoms, thesubstance exhibiting a binding affinity for at least wild type MurGenzyme and provided that the substance is not Lipid I, the naturalsubstrate of wild type MurG enzyme. More particularly, the substance ofthe invention serves as an acceptor for the GlcNAc transferase activityof at least wild type MurG enzyme or its homologs.

Also disclosed is a method of detecting GlcNAc transferase activity in asample suspected of containing a protein or an active fragment thereofexhibiting GlcNAc transferase activity. Preferably the method comprises(a) providing a sample suspected of containing a protein or an activefragment thereof exhibiting GlcNAc transferase activity; (b) contactingthe sample with effective amounts of labeled GlcNAc substrate and asubstance comprising the chemical moiety of the formula (I), above,under conditions effective to provide a labeled coupling productcomprising labeled GlcNAc coupled to the substance via a glycosidic bondin the presence of a protein or an active fragment thereof exhibitingGlcNAc transferase activity; and (c) detecting the formation or presenceof the labeled coupling product, which is indicative of GlcNActransferase activity in the sample.

It is also an objective of the present invention to provide an assay fordetecting GlcNAc transferase activity in a sample suspected ofcontaining a protein or an active fragment thereof exhibiting GlcNActransferase activity comprising a compound of the formula (I), above. Ascreen and methods of utilizing same are also contemplated by thepresent invention. In particular, a screen is provided for compoundsexhibiting potential antibacterial activity comprising (i) a protein oran active fragment thereof exhibiting GlcNAc transferase activity, (ii)a substance comprising the chemical moiety of the formula (I), above,and (iii) a labeled GlcNAc substrate.

Additionally, the method of this invention provides a detection stepcomprising binding the “A” or “R₃” groups of formula 1 to a solidsupport via a biotin tag, wherein said solid support includes an avidinor streptavidin coated resin. This step provides a continuous monitoringof product formation via the use of scintillation proximity assay.Furthermore, the separation of biotin-labeled substance involvesfiltration through an avidin-coated resin.

In a preferred embodiment of the invention “R₃” may be selected from H,an aliphatic group comprising 1 to about 50 carbon atoms, an aromatic orheteroaromatic group comprising 3 to about 55 carbon atoms,pyrophosphate protecting groups and pharmaceutically acceptable saltsthereof.

Additionally, a method detection step comprises binding said “A” or “R₃”to a solid support via a biotin tag, wherein said solid support includesan avidin or streptavidin coated resin.

Hence, substrate analogs are prepared, which are used in an enzyme assayfor MurG or MurG-like activity. A direct assay for MurG activity is thusprovided.

These and other objects of the invention are described further, below,along with the preferred embodiments of the invention.

4. BRIEF DESCRIPTION OF THE FIGURE

FIG. 1. Plot of GlcNAc transfer as a function of the concentration ofsubstrate analog 5b and concentration of active MurG enzyme. Allreactions are run in 100 mM Tris-HCl, pH 7.6, 1 mM MgCl₂, with 0.5-1.0μg total protein and 9.4 μM 14C-UDP-GlcNAc (265 mCi/mmol). Reactions forcurves A, B, C, and D are carried out using a cell lysate from atransformed BL21(DE3)pLysS strain that overexpresses MurG: A)-▪7.1 μM5b; B)-♦3.5 μM 5b; C)-0.71 μM 5b; D)-◯7.1 M 5b+heat treated cell lysate(65° C., 5 min.). Reactions for curve E are carried out using aBL21(DE3)plysS cell lysate expressing only endogenous levels of MurG:E)-∇7.1 μM 5b.

FIG. 2(a). Double reciprocal plots of the initial rate data withUDP-GlcNAc as the varied substrate. Initial rates are measured at fixedacceptor 1 b concentrations of 7 μM (⋄), 10 μM (∇), 15 μM (▪), 30 μM(+), 100 μM (). 0.08 μM of purified MurG is used for each reaction.

FIG. 2(b). Secondary plots of the slope.

FIG. 2(c). Intercept versus [1b]⁻¹. Analysis of the data assuming arapid equilibrium sequential mechanism yields the following kineticparameters: K_(UDP-GLCNAC)=110±+uM K_(1b)=60±15 μM.

FIG. 3. IC₅₀ measurements for compound 12a and UDP. All the assays areperformed under the same conditions with 18 μM 1b and 34.3 μMUDP-GlcNAc. Each IC₅₀ value is determined by fitting five or six datapoints to equation:$\frac{v_{i}}{v_{o}} = \frac{1}{1 + \frac{(1)}{{IC}_{50}}}$

where v_(i) is the initial rate in the presence of inhibitor atconcentration (1), and v_(o) is the initial rate without inhibitor.

FIG. 4. Structure of Lipid I and analogs (1a, 1b).

FIG. 5. Substrate-based inhibitors of MurG activity.

FIG. 6. Alternative acceptors for MurG.

FIG. 7. Synthesis of lipid I analogs (1a, 1b). Reagents and conditions:(a) CCl₃CH₂OH, DCC/DMAP, THF, rt, 4 h, 80%; (b) 1. H₂/Pd, EtOAc, rt, 0.5h; 2. PhCH(OCH₃)₂, cat TsOH, DMF, rt, 10 h, 81%, 2 steps; (c)iPr₂NP(OBn)₂, ¹H-tetrazole, CH₂Cl₂, −20° C.->0° C., 0.5 h, then mCPBA,−40° C.->25° C., 2 h, 70%; (d) Zn dust, 90% AcOH/H₂O, rt, 1 h, 91%; (e)HOBt, PyBop, DIEA, DMF, 0° C., 30 min, 87%; (f) 1. H₂/Pd, CH₃OH, rt, 30min, then py; 2. (R)-(+)-β-Citronellol-OPO₃PO(OPh)₂, py, CH₂Cl₂, rt, 18h, 68%; (g) TBAF, DMF, rt, 24 h, 93%; (h) 6-((biotinoyl)amino)hexanoicacid succinimide ester, NaHCO₃, H₂O/dioxane, rt, 2 h, 80%.

FIG. 8. Synthesis of disaccharide by MurG.

5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

5.1. General Aspects of the Invention

The present invention contemplates a substance comprising the chemicalmoiety of the formula:

in which “R” is an acyl group comprising 2 or more carbon atoms, “R₁” isa substituted or unsubstituted alkyl group comprising 1 or more carbonatoms, “R₂” is hydrogen or a substituted or unsubstituted alkyl groupcomprising 1 or more carbon atoms, “A” is a substituted or unsubstitutedamino acid residue or a peptide comprising 2 or more substituted orunsubstituted amino acid residues and “R₃” is a substituted orunsubstituted alkyl group comprising 5 or more carbon atoms, such as 15to 40 carbon atoms or 10 to 40 carbon atoms based on citronellolcontaining 10 carbon atoms. Preferably, the substance of the invention(sometimes referred to herein as a substrate analog or, simply,compound) exhibits a binding affinity for at least wild type MurGenzyme. More preferably, the substance of the invention serves as anacceptor for the GlcNAc transferase activity of at least wild type MurGenzyme. It is important to note that the substance of the invention isnot so broadly defined as to encompass Lipid I, the natural substrate ofwild type MurG enzyme.

It should be evident to one of ordinary skill that the substancedisclosed and described herein can also possess inhibitory activityagainst the GlcNAc transferase activity of at least wild type MurGenzyme, its homologs and, possibly, certain mutant forms thereof,depending in part on the strength of its binding affinity with theprotein or its active fragments. That is, a substrate analog of thepresent invention, by binding tenaciously to the protein or activefragment thereof, can potentially inhibit the ability of MurG or aMurG-like enzyme to catalyze the glycosylation reaction that results inthe transfer of GlcNAc to the C4 hydroxyl position of theN-acetylmuramic acid moiety of Lipid I. Of course, MurG and its homologsare derived from E. coli and other gram-negative bacteria. Gram-positivebacteria, such as B. subtilis, E. faecalis, E. hirae, as well as M.tuberculosis, are also known to harbor homologs of MurG.

Accordingly, in a preferred embodiment of the invention, “R” is an acylgroup including, but not limited to, acetyl, proprionyl, butanoyl,pentanoyl, hexanoyl and the like. The group “R₁” is a substituted orunsubstituted alkyl group including, but not limited to, methyl, ethyl,propyl, butyl, pentyl, hexyl, phenyl, benzyl, tolueyl, anthracyl and thelike. The group “R₂” is a hydrogen or a substituted or unsubstitutedalkyl group including, but not limited to, methyl, ethyl, propyl, butyl,pentyl, hexyl, phenyl, benzyl, tolueyl, anthracyl and the like.

Hence, the term “alkyl” group can encompass an aliphatic or an aromaticgroup, and the term “substituted” means that the particular alkyl groupcan have substituents including, but not limited to, additional alkylgroups, heteroatoms or functional groups containing heteroatoms,including, but not limited to, alcohols, ethers, carboxylic acids,esters, amides, amines, alkylamines, thiols, sulfides, sulfates,sulfoxides, sulfonic acids, phosphoric acids, phosphate esters,phosphides, phosphonates, phosphoramidates and the like. Any acyl groupcan have 2 or more carbon atoms, and any alkyl group can have 1 or morecarbon atoms. Each group can have as many as 25 carbon atoms, preferablyup to 20 carbon atoms, more preferably up to 15, most preferably up to10 carbon atoms.

In one embodiment of the invention, the group “R₃” may be a substitutedor unsubstituted alkyl group including, but not limited to, methyl,ethyl, propyl, butyl, pentyl, hexyl, phenyl, benzyl, tolueyl, naphthyl,anthracyl and the like. More particularly, the group “R₃” comprises amimic of the 55-carbon hydrocarbon anchor found in the natural MurGsubstrate, Lipid I. Such mimics include, but are not limited to,citronellol, other polyprenol derivative, or an aromatic group. Inaddition, the group “R₃” can be bound to a solid support, such as asynthetic resin or bead.

The group “A” is broadly contemplated to encompass any substituted orunsubstituted amino acid residue or any peptide comprising 2 or moresubstituted or unsubstituted amino acid residues. The group “A” can haveas few as one, two, or three amino acid residues, or many as 10 or moreamino acid residues, preferably no more than ten, more preferably nomore than eight, most preferably no more than five (e.g., apentapeptide). Other chemical moieties may be associated with the group“A,” preferably covalently attached, including but not limited to linkergroups, labeling groups (such as radiolabeled groups, fluorescent groupsand the like), affinity groups (such as biotin, avidin, streptavidin andthe like or haptens, such as dinitrophenol, digoxegenin and the like),hydrophobic groups, hydrophilic groups, and the like. In one embodimentof the invention biotin is conjugated to the group “A”, in which thebiotin moiety is attached covalently (e.g., to an amino group of anamino acid residue) either directly or via a linker moiety.

In a preferred embodiment, the amino acid residue attached to the lacticacid moiety of the substance of the formula (I) is Ala. A D-γ-linkedglutamic acid residue is preferably attached next to this first alanineresidue. A lysine residue (L-Lys) is preferably attached next to thisglutamic acid residue, particularly for gram-positive bacteria. Forgram-negative bacteria, this third residue is preferablymeso-diaminopimelate or “m-DAP.” Other residues at this positioninclude, but are not limited to, L-alanine, L-homoserine,L-diaminobutyric acid, L-glutamic acid, L-ornithine, LL-DAP, as well asthe meso-form, referred to, above. Still others may include L-Orn,LL-Dpm, m-HyDpm, L-Dab, L-HyLys, N^(γ)-Acetyl-L-Dab, L-Hsr, L-Ala, orL-Glu. A preferred amino acid sequence for a pentapeptide isL-Ala-D-γ-Glu-L-Lys-D-Ala-D-Ala, the amino terminal end of which isattached to the lactic acid moiety of the substance of the formula (I)via an amide bond. Yet another suitable amino acid sequence may beL-Ala-D-γ-Glu-meso-DAP-D-Ala-D-Ala. A tripeptide sequence of potentialadvantage is L-Ala-D-γ-Glu-L-Lys, optionally substituted at the L-Lysamino acid residue with an affinity “handle,” such as biotin, avidin,streptavidin, an immunoglobulin, Protein A, and the like or fragmentsthereof, or haptens, such as dinitrophenol, digoxegenin and the like.Still possible is a dipeptide arrangement, including but not limited toL-Ala-D-Lys, once again optionally substituted at the D-Lys amino acidresidue.

In a method of the present invention GlcNAc transferase activity isdetected in a sample suspected of containing a protein or an activefragment thereof exhibiting GlcNAc transferase activity. The methodincludes the steps of: (a) providing a sample suspected of containing aprotein or an active fragment thereof exhibiting GlcNAc transferaseactivity; (b) contacting the sample with effective amounts of labeledUDP-GlcNAc substrate and a substance comprising the chemical moiety ofthe formula (I), above, provided that the substance is not Lipid I, thenatural substrate of wild type MurG enzyme, under conditions effectiveto provide a labeled coupling product comprising labeled GlcNAc coupledto the substance via a glycosidic bond in the presence of a protein oran active fragment thereof exhibiting GlcNAc transferase activity; and(c) detecting the formation or presence of the labeled coupling product,which is indicative of GlcNAc transferase activity in the sample.Preferably, the labeled GlcNAc substrate is labeled UDP-GlcNAc.

In the inventive method at least a portion of the sample may comprise aportion of a lysed bacterial culture, a portion of a supernatantthereof, a portion of a membrane fraction thereof, a portion of aprotein fraction thereof, a purified enzyme, purified or synthesizedlipid or mixtures of same.

Detection of the formation or presence of the labeled coupling productcan be effected in a number of ways, apparent to those of ordinaryskill. For example, the detection step may comprise separation of thelabeled coupling product from labeled UDP-GlcNAc substrate. As discussedelsewhere in this disclosure, separation of the labeled species can beaccomplished using a variety of approaches, including but not limitedto, hydrophobic capture, affinity chromatography, or other solid phaseseparation techniques. Quantification of the labeled coupling productcan then follow depending on the nature of the label utilized.

Consistent with the objectives of the present invention an assay isprovided for detecting GlcNAc transferase activity in a sample suspectedof containing a protein or an active fragment thereof exhibiting GlcNActransferase activity. An assay of the invention comprises a compound ofthe formula:

in which “R” is an acyl group comprising 2 or more carbon atoms, “R₁” isa substituted or unsubstituted alkyl group comprising 1 or more carbonatoms, “R₂” is a hydrogen or a substituted or unsubstituted alkyl groupcomprising 1 or more carbon atoms, “A” is a substituted or unsubstitutedamino acid residue or a peptide comprising 2 or more substituted orunsubstituted amino acid residues, “R₃” is a substituted orunsubstituted alkyl group comprising 5 or more carbon atoms, thesubstance able to form a coupling product with a GlcNAc substrate in thepresence of a protein or an active fragment thereof exhibiting GlcNActransferase activity, provided that the substance is not Lipid I, thenatural substrate of wild type MurG enzyme. The assay further comprisesa labeled GlcNAc substrate.

A screen for compounds exhibiting potential antibacterial activity isalso contemplated. Such a screen comprises: (i) a protein or an activefragment thereof exhibiting GlcNAc transferase activity, (ii) asubstance comprising the chemical moiety of the formula (1), above, thesubstance able to form a coupling product with a GlcNAc substrate in thepresence of a protein or an active fragment thereof exhibiting GlcNActransferase activity, provided that the substance is not Lipid I, thenatural substrate of wild type MurG enzyme, and (iii) a labeled GlcNAcsubstrate.

Thus, a screen including the enzyme MurG, or an active fragment thereof,is brought into contact with a substrate analog, such as a substance ofthe formula (I), in the presence of labeled GlcNAc substrate. Theenzyme, of course, would catalyze the coupling of the labeled GlcNAc(e.g., from C-14 labeled UDP-GlcNAc) to the C4-hydroxyl group of themuramic acid moiety of the substrate analog. The formation of labeledcoupling product is then monitored over time to produce a graph, such asthat presented in FIG. 1. (The coupling product may first have to beseparated from labeled GlcNAc substrate, e.g., by column chromatography,HPLC, filtration (if the reaction is conducted in the solid phase) andthe like.) A potential inhibitory compound (or compounds) is then addedto the mixture, such as the control mixture described above, and thedecrease in the production of labeled coupling product is monitored,preferably as a function of the concentration of the potentialinhibitory compound.

5.2. The Preparation of a Substrate Analog

Our first synthetic target, 8 (above, and Scheme 2, below), differs fromLipid I in that the 55 carbon undecaprenol chain has been replaced bythe ten carbon chain of citronellol. A shorter lipid chain is chosenbecause long chain lipids are difficult to handle; a lipid containing asaturated isoprenol unit is further chosen because allylicpyrophosphates are unstable. Although MurG is a membrane-associatedenzyme, which recognizes a lipid-linked substrate, the chemistry takesplace on the C4 hydroxyl of the lipid-linked substrate, which is farremoved from the lipid anchor. Therefore, it is hoped that alteration ofthe lipid can be accomplished without destroying substrate recognition.

To make 8 (See, Scheme 2, below), muramic acid derivative 1 (availablefrom Sigma) is converted to the anomeric dibenzyl phosphate 5 in 5 stepsand coupled to the protected pentapeptide 13. Chen, J.; Dorman, G.;Prestwich, G. J. Org. Chem. 1996, 61, 393. The silyl protecting groupson the Lys and Glu are preferred for facile deprotection under mildconditions. Hence, the C-terminus of the peptide can be a methyl ester,as shown, or a trimethylsilyl ethyl ester.

The protected pentapeptide is synthesized on a D-Ala-FMOC Sasrin resin(available from Bachem Biosciences) in 11 steps in an overall yield of15% (See, Method 4, below). Experimental details are provided in theExamples Section, below. Hydrogenolytic deprotection produces theanomeric phosphate, which is treated with diphenyl citronellolpyrophosphate to produce 7 (See, Scheme 2). Diphenyl citronellolpyrophosphate (10, Method 1, below) is generated in situ by treatingcitronellol phosphate with diphenyl chlorophosphate (See, ExampleSection, below; see, also: Warren, C. D.; Jeanloz, R. W. Meth. Enzymol.1978, 50, 122.) For other methods to form glycosyl pyrophosphates, see:(a) Imperiali, B.; Zimmerman, J. W. Tet. Lett. 1990, 45, 6485; (b)Wittmann, V.; Wong, C.-H. J. Org. Chem. 1997, 62, 2144. Thepyrophosphate exchange reaction takes place readily in the presence ofthe unprotected sugar hydroxyls. Finally, the side chain protectinggroups on the peptide are removed with TBAF, which also hydrolyzes theC-terminal methyl ester to give the desired product 8. It should benoted that 8 is both acid- and base-sensitive. The synthesis minimizesexposure, to acid and base, while providing for a convergent approachthat allows independent modification of all three building blocks, thepeptide, the carbohydrate, and the lipid.

Thus, using the same general scheme described above and furtherillustrated below (in which TMSE is trimethylsilylethyl, TEOC istrimethylsilylethyloxycarbonyl and N-linker is 6-aminohexanoic acid),one can prepare a variety of compounds to define the requirements forsubstrate binding.

5.3. GlcNAc Transferase Assay

Initial attempts to use substrate 8 in MurG activity assay reveals someInitial attempts to use substrate 8 in MurG activity assay reveals somedifficulties in separating radiolabeled product from excess labeledUDP-GlcNAc, using relatively crude separation methods like paperchromatography or thin layer chromatography. Hence, in certainapplications, it may be preferable to adjust the length of the lipidchain to facilitate removal of excess labeled UDP-GlcNAc. For instance,a longer lipid chain (e.g., ca C₁₅-C₄₀) may facilitate a separationmethod using a hydrophobic resin or suitable filter to take advantage ofnon-specific lipid-lipid interactions. What is more, a tether to a solidphase resin may be more preferable in a commercial embodiment of theinvention. Still another alternative comprises an affinity group, suchas biotin, an IgG binding domain, or a hapten, such as dinitrophenol,digoxegenin and the like, which is attached to the substrate analog tofacilitate separation by affinity chromatography using an affinity resincomprising avidin/streptavidin or

The evidence suggests that MurG is relatively insensitive to theidentity of the third amino acid residue in the peptide chain. E. colistrains (e.g., BL21) make a muramyl pentapeptide substrate withmeso-diaminopimelic acid (m-DAP) rather than L-lysine. E. coil MurGaccepts these lysine analogs. Fluorescently labeled analogs are alsoaccepted coil MurG accepts these lysine analogs. Fluorescently labeledanalogs are also accepted by some strains: Weppner, W. A.; Neuhaus, F.C. J. Biol. Chem. 1978, 253, 472; White, D. Physiology and Biochemistryof Prokaryotes Oxford Univ. Press:New York, 1995, pp 212-223.Accordingly, the third amino acid residue makes a convenient locationfor attaching substituents onto the amino acid/peptide moiety. In apreferred embodiment of the invention, an affinity label substitutedL-Lys is used as the third amino acid residue of the peptide chain. Morepreferably a biotin moiety is linked to the free amino group of lysinevia a tether comprising a bifunctional aliphatic agent, such as6-aminohexanoic acid, although shorter or longer tethers can be used.Tethers of various lengths, which are attached to certain molecules ofinterest, such as biotin, chromophores, fluorophores and

In this manner, biotin is attached (Scheme 2)(6-{(biotinoyl)amino}hexanoic acid succinimide ester can be purchasedfrom Molecular Probes, Inc.) to the ε amino group of the lysine residuevia the carboxylic acid group of the 6-aminohexanoic acid linker so thatreaction mixture using an avidin-derivatized resin (Tetralink™Tetrameric Avidin Resin, Promega). The ability of MurG to recognize thebiotin-labeled substrate 9 is evaluated by membrane preparations with 9and ¹⁴C-UDP-GlcNAc. (See, e.g., Baker, C. A.; Poorman, R. A.; Kezdy, F.J.; Staples, D. J.; Smith, C. W.; Elhammer, A. P. Anal. Biochem. 1996,239, 20.) The reaction is rapid and efficient with a bacterial culturethat overexpresses MurG but barely detectable with a culture expressingonly endogenous levels of MurG (FIG. 1; compare curves A and E).

The murG gene can be obtained from the pUG18 plasmid available fromProf. W. D. Donachie (Univ. of Edinburgh). The E. coli murG genesequence is described by Ibid. 1990, 18, 4014. Gene amplification bypolymerase chain reaction using the pUG18 plasmid as the template isperformed. The pT7BlueT PCR cloning vector, which is available fromNovagen, is used for this purpose. The DNA fragment that contains murGis cleaved from pT7BlueT plasmid by restriction enzymes NdeI and BamHI,and the NdeI/BamHI cloning site of the pET15b expression vector, alsoavailable from Novagen.

The murG gene is subcloned from pET15b into a pET3a plasmid (Novagen).MurG is overexpressed in the IPTG-inducible BL21(DE3)pLysS strain(Novagen). See: Studier, F. W.; Rosenberg, A. H.; Dunn, J. J.;Dubendorff, J. W. Meth. Enzymol. 1990, 185, 60. Heat treating theoverexpressing cell lysate prior to adding it to the substrates preventsthe reaction from proceeding (See, FIG. 1; compare A and D). Hence, thereaction depends on the presence of active MurG. Furthermore, both theinitial reaction rate and conversion to coupled product increases withthe concentration of 9 (See, FIG. 1; compare A, B. and C).

Therefore, the synthetic substrate analog functions efficiently in adirect assay for MurG activity despite having a different, anddramatically shorter, lipid chain. This synthetic substrate can be usedto evaluate enzyme activity in overexpressing cell lysates, followingstructural modifications to the murG gene which produce amino acidtruncations, additions, deletions, substitutions, or other mutations.The synthetic substrate analog can also be used to assay for enzymeactivity during purification, as well as for detailed mechanisticstudies on wholly or partially purified enzyme. Thus, a high resolutionstructural analysis of MurG is now possible. In addition, by evaluatingthe ability of other synthetic substrates to compete with 9 for¹⁴C-UDP-GlcNAc, it is possible to identify simpler acceptors for use indirect screens for MurG inhibition.

As a further illustration of the invention, the following examples areprovided.

6. EXAMPLES

The following procedures are provided making specific reference toScheme 2, above, and Methods 1-4, below.

6.1. Preparation of Compound 2

Compound 1 (482 mg, 1.022 mmol, Sigma) and 4-dimethylaminopyridine (10mg, 0.080 mmol) are premixed, dried three times by azeotropicdistillation with toluene and then dissolved in 8 mL of tetrahydrofuran(THF). Trichloroethanol (0.23 mL, 2.405 mmol) is added to the reactionvessel followed by 1,3-dicyclohexylcarbodiimide (248 mg, 1.203 mmol).After sitting at room temperature for 4 h, the reaction solution isfiltered through a cotton plug and rinsed with ethyl acetate (EtOAc).The filtrate is concentrated and purified by flash chromatography (15%EtOAc/CH₂Cl₂) to give 453 mg (80%) of a white powder. R_(f)0.39 (15%EtOAc/ CH₂Cl₂); ¹H NMR (CDCl₃, 270 MHz) δ7.43-7.25(m, 10 H), 7.67(d,J=6.0 Hz, 1 H), 5.59 (s, 1 H), 5.34 (d, J=3.2 Hz, 1 H), 4.98 (d, J=11.9Hz, 1 H), 4.71-3.70 (m, 10 H), 2.04 (s, 3 H), 1.50 (d, J=7.0 Hz, 3 H);Mass spec. [M+H]⁺, 603.5.

6.2. Preparation of Compound 3

Compound 2 (360 mg, 0.599 mmol) is dissolved in 30 mL of EtOAc, and 900mg of 20% Pd—C is added. The reaction vessel is filled with hydrogen andstirred at room temperature. After 30 min, the catalyst is filtered offand washed with methanol. The filtrate is concentrated to give a fullyhydrogenated product, which is used in the next

To a solution of the triol in 6 mL of DMF is added benzylaldehydedimethyl acetal (0.9 mL, 6 mmol) and p-toluenesulfonic acid (11.4 mg,0.06 mmol). The reaction mixture is stirred at room temperature for 10h, neutralized with saturated NaHCO₃, extracted with CH₂Cl₂ (3×20 mL),dried over Na₂SO₄, filtered, concentrated, and purified by flashchromatography (90% EtOAc/petroleum ether) to give 248 mg (81%) of amixture of α, β anomers.

6.3. Preparation of Compound 4

Compound 3 (202 mg, 0.395 mmol) and 1H-tetrazole are premixed and driedby azeotropic distillation with toluene, then dissolved in 10 mL ofCH₂Cl₂, and cooled to −30° C. To the solution is added dibenzylN,N-diisopropylphosphamide (0.266 mL, 0.791 mmol). The mixture isstirred at room temperature for 1 h and cooled to −40° C., m-CPBA (560mg, 2 mmol) is added, and the reaction is stirred for 30 min at 0° C.and then 30 min at room temperature. The mixture is diluted with CH₂Cl₂,washed with 10% aqueous Na₂SO₃, saturated NaHCO₃, and water; then driedover Na₂SO₄, filtered, concentrated, and purified by flashchromatography (65% EtOAc/petroleum ether) to give 200 mg (70%) of whitesolid. R_(f)0.24 (70% EtOAc/petroleum ether); ¹H NMR (CDCl₃, 270 MHz)δ7.44-7.33 (m, 15 H), 7.20 (d, J=6.0 Hz, 1 H), 6.10 (m, 1 H), 5.56 (s, 1H), 5.05 (m, 6 H), 4.61 (q, J=7.0 Hz, 2 H), 4.10-3.61 (m, 6 H), 1.86 (s,3 H), 1.48 (d, J=7.0 Hz, 3 H).

6.4. Preparation of Compound 5

Zinc dust is added to a solution of compound 4 (58 mg, 0.0752 mmol) in 5mL of 90% AcOH/H₂O. The mixture is stirred vigorously at roomtemperature. After 1 h, the catalyst is filtered off, the filtrate isconcentrated and purified by flash chromatography (10% MeOH/CHCl₃, 0.1%AcOH) to give 44 mg (91%) of product. R_(f)0.19 (5% MeOH/CHCl₃, 0.1%AcOH); ¹H NMR (CD₃OD, 500 MHz) δ7.44-7.25 (m, 15 H), 6.11 (m, 1 H), 5.55(s, 1 H), 5.02 (m, 4 H), 4.33 (q, J=7.0 Hz, 1 H), 3.96 (m, 1 H), 3.77(m, 1 H), 3.73-3.66 (m, 4 H), 1.94 (s, 3 H), 1.32 (d, J=7.0 Hz, 3 H).

6.5. Preparation of Compound 6

Compound 5 (45 mg, 0.0704 mmol) andNH₂-L-Ala-γ-D-Glu(O-TMSE)-L-Lys(N-TEOC)-D-Ala-D-Ala-OCH₃ (35 mg, 0.0469mmol) are premixed and dried by azeotropic distillation with toluenethree times, then dissolved in 0.9 mL of DMF, then cooled to 0° C.Diisopropylethylamine (41 μL, 0.235 mmol) is added to reaction vesselfollowed by HOBt (12.7 mg, 0.0938 mmol) and pyBOP (49 mg, 0.0938 mmol).After stirring for 30 min at room temperature, the solution is dilutedwith 10 mL of EtOAc, washed with 0.01 N aqueous HCl, and water. Thesolution is then concentrated and purified by flash chromatography (5%MeOH/CHCl₃) to give 59 mg (92%) of compound 6. R_(f)0.16 (5%MeOH/CHCl₃); ¹H NMR (CD₃OD, 500 MHz) δ7.50-7.30 (m, 15 H), 5.87 (m, 1H), 5.63 (s, 1 H), 5.13 (m, 4 H), 4.41 (m, 1 H), 4.40 (m, 1 H), 4.36 (m,1 H), 4.35 (m, 1 H), 4.30 (m, 1 H), 4.21 (m, 1 H), 4.19 (m, 2 H), 4.14(m, 2 H), 4.13 (m, 1 H), 4.05 (m, 1 H), 3.84 (m, 1 H), 3.79 (m, 2 H),3.76 (m, 1 H), 3.66 (s, 3 H) ,3.09 (t, J=8.8 Hz, 2 H), 2.28 (t, J=8.8Hz, 2 H), 2.18 (m, 1 H), 1.91 (m, 1 H), 1.86 (s, 3 H),177 (m, 1 H), 1.67(m, 1 H), 1.51 (m, 2 H), 1.43-1.35 (m, 18 H), 1.01-0.97 (m, 4 H),0.05-0.02 (s, s, 18 H); Mass spec [M+H]⁺ 1394.

6.6. Preparation of Compound 7

Compound 6 (15 mg, 0.011 mmol) is dissolved in 1 mL of MeOH and 20 mg of20% Pd—C is added. The reaction vessel is filled with hydrogen andstirred at room temperature. A drop of diisopropylethylamine is addedafter 30 min, then the solution is diluted in 5 mL of MeOH and stirredfor 20 min. The mixture is filtered, concentrated to give thehydrogenated, debenzylated product (7a), which is used in the nextreaction without further purification. R_(f)0.28(CHCl₃:MeOH:H₂O=3:2:0.5).

Citronellol phosphate (diisopropylethylammonium, 18 mg, 0.053 mmol) isdried three times by azeotropic distillation with toluene, thendissolved in 1 mL of CH₂Cl₂. Diisopropylethylamine (18.5 μL, 0.106 mmol)is added. The solution is cooled to −20° C., anddiphenylphosphorochloridate (11.5 μL, 0.080 mmol) is added. The reactionvessel is allowed to warm up to room temperature and stirred for 1 h atroom temperature. After the addition of methanol (0.1 mL), the reactionis stirred for a further 1 h at room temperature, then the solvents areevaporated, and the residue is dried twice by azeotropic distillationwith toluene and dissolved in 0.2 ml of DMF.

Compound 7a from above is dried three times by azeotropic distillationwith toluene and dissolved in 0.1 mL of DMF. Diisopropylethylamine (3.9μL, 0.022 mmol) is added. 0.1 mL of the citronelloldiphenylpyrophosphate solution is transferred to the solution containingcompound 7a. The reaction mixture is stirred for 48 h at roomtemperature, then loaded directly to a C18 reverse phase column (8 mm×80mm, particle size 40 μm, pore size 60 Å, from J. T. Baker) and elutedwith CH₃CN/H₂O (0, 5%, 10%, 15%, 20%, 25%, 30%, 35% of 10 mL each) with0.1% triethylamine. The fractions containing the pure compound arecombined and concentrated to give 4.6 mg (28%) of white powder.R_(f)0.36 (CHCl₃:MeOH:H₂O=3:2:0.5); ¹ H NMR (DMSO, 500 MHz) δ8.36 (d,J=7.2 Hz, 1 H), 8.21 (d, J=8.0 Hz, 1 H), 8.19 (d, J=8.2 Hz, 1 H), 8.10)d, J=6.0 Hz, 1 H), 7.32 (d, J=7.5 Hz, 1 H), 6.95 (t, J=5.0 Hz, 1 H),5.26 (d, J=6.0 Hz, 1 H), 5.07 (t, J=7.0 Hz, 1 H), 4.30 (m, 1 H), 4.27(m, 1 H), 4.23 (m, 1 H), 4.13 (m, 1 H), 4.12 (m, 2 H), 3.87 (m, 1 H),3.77 (m, 2 H), 3.62 (m, 1 H), 3.60 (s, 3 H), 3.51 (m, 1 H), 3.33 (m, 1H), 2.91 (m, 2 H), 2.17 (m, 2 H), 1.94 (m, 2 H), 1.91 (m, 1 H), 1.80 (s,3 H), 1.62 (s, 3 H), 1.58 (s, 3 H), 1.51 (m, 3 H), 1.50 (m, 1 H), 1.49(m, 1 H), 1.35 (m, 2 H), 1.29 (d, j=7.2 Hz, 3 H), 1.27 (m, 2 H, 1.25 (d,J=6.8 Hz, 3 H), 1.24 (d, J=5.5 Hz, 3 H), 1.23 (m, 2 H), 1.19 (d, J=7.4Hz, 3 H), 1.11 (m, 1 H), 0.84 (d, J=6.5 Hz, 3 H), 0.02-0.01 (s, s, 18H); Mass spec. [M+H]⁺ 1321.

6.7. Preparation of Compound 8

To a solution of compound 7 (5 mg, 0.0033 mmol) in 50 μL of DMF is addedtetrabutylammonium fluoride (1 M in THF, 0.3 mL). The reaction mixtureis stirred for 24 h at room temperature, then loaded directly to a C18reverse phase column (8 mm×80 mm, particle size 40 μm, pore 60 Å, fromJ. T. Baker) and eluted with CH₃CN/0.1% NH₄HCO₃ aqueous solution (0, 5%,10%, 15%, 20%, 25%, 30% of 10 mL each). The fractions containing thepure compound are combined, concentrated, and lyophilized to removesalts. A white powder (2 mg, 57%) is obtained. R_(f)0.18(CHCl₃:MeOH:H₂O=3:3:1); ¹H NMR (CD₃OD, 500 MHz) δ5.58 (m, 1 H), 5.11 (t,J=6.5 Hz, 1 H), 4.50-3.56 (m, 12 H), 2.94 (m, 2 H), 2.34 (m, 2 H), 2.10(s, 3 H), 2.00 (m, 1 H), 1.98 (m, 2 H), 1.92 (m, 1 H), 1.74 (m, 2 H),1.67 (s, 3 H), 1.62 (m, 1 H), 1.60 (s, 3 H), 1.50-1.39 (m, 12 H), 1.23(m, 2 H), 0.93 (d, J=6.5 Hz, 3 H); Mass spec. [M+H]⁺ 1062.

6.8. Preparation of Compound 9

To a solution of compound 8 (2 mg, 0.0019 mmol) in 0.1 mL ofH₂O/dioxane(1:1) is added NaHCO₃ (3.2 mg, 0.038 mmol), followed by6-((biotinoyl)amino)hexanoic acid succinimide ester (2 mg, 0.0044 mmol).The reaction mixture is stirred for 2 h at room temperature, then loadeddirectly to a C18 reverse phase column (8 mm×80 mm, particle size 40 μm,pore size 60 Å, from J. T. Baker) and eluted with CH₃CN/0.1% NH₄HCO₃aqueous solution (0, 5%, 10%, 15%, 20%, 25%, 30% of 10 mL each). Thefractions containing the pure compound are combined, concentrated, andlyophilized to remove salts. A white powder (2 mg, 76%) is obtained.R_(f) 0.40 (CHCl₃:MeOH:H₂O=3:3:1); ¹H NMR (CD₃OD, 500 MHz) δ5.52 (d,J=4.5 Hz, 1 H), 5.12 (t, J=7.0 Hz, 1 H), 4.50 (m, 1 H), 4.39-4.19 (m, 8H), 4.00-3.72 (m, 4 H), 3.51 (m, 1 H), 3.22 (m, 1 H), 3.18 (m, 2 H),2.95 (dd, J=12.5, 5.0 Hz, 1 H), 2.71 (d, J=12.5 Hz, 1 H), 2.27 (m, 2 H),2.02 (s, 3 H), 2.01 (m, 2 H), 1.85 (m, 2 H), 1.67 (m, 2 H), 1.67 (s, 3H), 1.62 (m, 1 H), 1.61 (s, 3 H), 1.53 (m, 2 H), 1.45-1.37 (m, 12 H),1.38 (m, 1 H), 1.17 (m, 1 H), 0.94 (d, J=6.8 Hz, 3 H); Mass spec. [M+H]⁺1402.

6.9. Preparation of Compound 10 (Method 1, below)

(R)-(+)-β-citronellol (330 mg, 2.111 mmol) is dried three times byazeotropic distillation with toluene, then dissolved in 21 mL of dryhexane. In another dry flask, phosphorus oxychloride (0.98 mL, 10.56mmol) and triethylamine (1.47 mL, 10.56 mmol) are dissolved in 10 mL ofdry hexane and stirred at room temperature. The citronellol solution isthen added slowly (over 1 h) to the phosphorus oxychloride solutionafter which stirring is continued for 30 min. A mixture of 70 mLacetone/water/triethylamine (88:10:2) is added to the reaction, which isallowed to stir for 18 h at room temperature to convert citronellolphosphate dichloride to citronellol phosphate. The solvent is evaporatedin vacuo to give an aqueous residue, which is loaded to a C18 reversephase column 50 mm×12 cm, particle size 40 μm, pore size 60 Å, from J.T. Baker) and eluted with CH₃CN/H₂O (0, 10%, 20%, 30%, 40%, 50% 60% of100 mL each). The fractions containing the pure compound are combinedand concentrated to give 566 mg (62%) of oily residue. R_(f)0.42(CHCl₃:MeOH:H₂O=3:2:0.5); ¹H NMR (CD₃OD, 500 MH) δ5.09 (t, J=5.0 Hz, 1H), 3.90 (m, 2 H), 1.99 (m, 2 H), 1.67 (m, 1 H), 1.65 (s, 3 H), 1.62 (m,1 H), 1.59 (s, 3 H), 1.41 (m, 1 H), 1.34 (m, 1 H), 1.16 (m, 1 H), 0.91(d, J=6.5 Hz, 3 H); ¹³C NMR (CD₃OD, 500 MHz) δ132.08, 125.96, 65.11,39.00, 38.94, 30.45, 26.61, 26.10, 19.93, 17.92.

Method 1

(a) 5 eq. POCl3, TEA, hexane, rt, 1 hr; then add acetone/H2O/TEA(85:10:5), 10 hrs, 70%.

Method 2

(a) 50% TFA/CH₂Cl₂, rt, 20 mins, 100%; (b) 1.2 eq.2-(Trimethylsilyl)ethyl p-nitrophenyl carbonate, DIEA, DMF, 50° C., 2hrs, 95%.

Method 3

(a) 1.2 eq. 9-Fluorenylmethyl chloroformate, 3 eq. NaHCO₃,H₂O/Dioxane(1:1), rt, 1 hr, 93% (b) 2 eq. trimethylsilyl ethanol,DCC/DMAP, EtOAc, rt, 2 hrs, 82%; (c) H2/Pd, MeOH, rt, 10 mins, 90%.

Method 4

*Sasrin resin-OOC-D-Ala-Fmoc, an acid-sensitive resin, is available fromBACHEM Bioscience Inc.

(a) 55% piperidine/NMP, rt, 30 mins; (b) 4 eq. HO-D-Ala-Fmoc, HOBT/HBTU,DIEA, NMP, rt, 2 hrs; identical coupling and deprotection conditions forHO-L-Lys(N-TEOC)-Fmoc, HO-D-γ-Glu(O-TMSE), and HO-L-Ala-Fmoc except forvariations in the amount of amino acid used; (c) 1% TFA/CH₂Cl₂, rt, 5×2mins; (d) KHCO₃, 50 eq. CH₃l, DMF, rt, 2 hrs overall yield 15%.

6.10. Preparation of Compound 11 (Method 2)

To a solution of OH-L-Lys(N-BOC)-NHFmoc (607 mg, 1.295 mmol) in 10 mL ofCH₂Cl₂ is added 10 mL of trifluoroacetic acid. The mixture is stirredfor 20 min at room temperature, then concentrated and lyophilized. Theresidue is dissolved in 10 mL of DMF, then diisopropylethylamine (1.13mL, 6.475 mmol) is added. 2-(Trimethylsilyl)ethyl p-nitrophenylcarbonate (440 mg, 1.554 mmol) is dissolved in 3 mL of DMF andtransferred into the L-Lys solution. The mixture is stirred for 2 h atroom temperature. The DMF solvent is evaporated in vacuo; the residue ispurified by flash chromatography (EtOAc, followed by 10% MeOH/CHCl₃ with0.1% AcOH) to give 635 mg (95%) of a white solid. R_(f)0.25 (10%MeOH/CHCl₃).

6.11. Preparation of Compound 12 (Method 3)

To a solution of D-Glu(benzyl) (1.046 g, 4.41 mmol) in 40 mL ofwater/dioxane (1:1) is added a solution of NaHCO₃ (1.1 g, 13.2 mmol) in10 mL of water. The mixture is stirred for 20 min. Then,9-Fluoenylmethyl chloroformate (1.37 g, 5.29 mmol) is dissolved in 10 mLof dioxane and added slowly (over 1 h) into the D-Glu solution afterwhich stirring is continued for 10 min. The mixture is loaded directlyto a silica gel column and eluted by 5% MeOH/CHCl₃ with 0.1% AcOH.Fractions containing product are combined, concentrated, and purifiedagain by flash chromatography (EtOAc, followed by 5% MeOH/CHCl₃ with0.1% AcOH) to give 1.88 g (93%) of a white powder, R_(f)0.27 (5%MeOH/CHCl₃ with 0.1% AcOH).

Fmoc-D-Glu(benzyl)-OH (350 mg, 0.762 mmol) and 4-dimethylaminopyridine(9.3 mg, 0.0762 mmol) are premixed and dried three times by azeotropicdistillation with toluene, and dissolved in 8 mL of EtOAc.Trimethylsilyl ethanol (0.328 mL, 2.287 mmol) is added to the reactionvessel followed by 1,3-dicyclohexylcarbodiimide (314 mg, 1.525 mmol).After stirring the mixture for 2 h at room temperature, the reactionsolution is filtered and washed with EtOAc. The filtration isconcentrated and purified by flash chromatography (15% EtOAc/petroleumether) to give 350 mg (82%) of a white powder. R_(f)0.33 (15%EtOAc/petroleum ether).

Fmoc-D-Glu(benzyl) 2-(trimethylsilyl) ethyl ester (270 mg, 0.483 mmol)is dissolved in 11 mL of methanol and 500 mg of 20% Pd-C is added. Thereaction vessel is filled with hydrogen and stirred at room temperature.After 10 min, the mixture is filtered, concentrated, and purified byflash chromatography (10% MeOH/CHCl₃) to give 203 mg (90%) of a whitepowder. R_(f)0.43 (10% MeOH/CHCl₃).

6.12. Preparation of Compound 13 (Method 4)

Sasrin resin-OOC-D-Ala-NHFmoc (800 mg, 0.56 mmol) is put in reactionvessel and washed successively by the following solvents (20 mL each):CH₂Cl₂ (2×3 min), N-methylpyrrolidone (NMP, 2×3 min), 20% piperidine/NMP(30 min), NMP (2×3 min), 50% dioxane/water (2×5 min), NMP (3×5 min),CH₂Cl₂ (3×3 min), NMP (1×3 min). OH-D-Ala-NHFmoc (701 mg, 2.24 mmol),diisopropylethylamine (0.59 mL, 3.36 mmol), HOBt/HBTU (0.45 M in DMF,2.5 mL), and 10 mL of NMP are added to the vessel and mixed thoroughly.The reaction vessel is shaken for 2 h at room temperature, then washedsuccessively with the following solvent (20 mL each): NMP (5×8 min),i-PrOH (5×8 min), CH₂Cl₂ (4×3 min) and NMP (2×3 min).

The same procedure is used for the other 3 amino acids except that theFmoc group is not cleaved for the last amino acid L-Ala-Fmoc.

After all of the amino acids are coupled, the pentapeptide is cleavedoff of the resin by washing with 1% TFA/CH₂CH₂ (5×2 mm, 15 mL each) withslight agitation. The cleavage solution is transferred via cannula intoa vessel containing 2 mL of pyridine and 20 mL of methanol. Thefiltration is concentrated and purified three times by flashchromatography (5% MeOH/CHCl₃ with 1% AcOH) to give 300 mg (56%) ofproduct. R_(f)0.34 (10% MeOH/CHCl₃).

KHCO₃ (28.4 mg, 0.284 mmol) is ground to a fine powder and mixed withFmoc-L-Ala-D-γ-Glu(O-TMSE)-L-Lys(N-TEOC)-D-Ala-D-Ala-OH (135.4 mg, 0.142mmol). The mixture is dissolved in 2 mL of DMF, CH₃I (0.44 mL, 7.1 mmol)is added. The mixture is stirred for 2 h at room temperature andpurified by flash chromatography (90% EtOAc/petroleum ether) to give 47mg (34%) of a white powder. R_(f)0.40 (100% EtOAc); ¹H NMR (DMSO-d₆, 500MHz) δ8.24 (d, J=7.4 Hz, 1 H), 8.18 (d, J=7.2 Hz, 1 H), 8.17 (d, J=8.4Hz, 1 H), 8.01 (d, J=7.1 Hz, 1 H), 7.89 (d, J=7.4 Hz, 2H), 7.72 (dd,J=7.4, 7.4 Hz, 2 H), 7.47 (d, J=7.8 Hz, 1 H), 7.41 (dd, J=7.4, 7.4 Hz, 2H), 7.32 (dd, J=7.4 7.4 Hz, 2 H), 6.94 (t, J=5.2 Hz, 1 H), 4.29-4.12 (m,8 H), 4.11 (t, J=8.8 Hz, 2 H), 4.00 (t, J=8.2 Hz, 2 H), 3.59 (s, 3 H),2.91 (m, 2 H), 2.17 (m, 2 H), 1.92 (m, 1 H), 1.79 (m, 1 H), 1.56 (m, 1H), 1.48 (m, 1 H), 1.35 (m, 2 H), 1.28 (d, J=7.3 Hz, 3 H), 1.23 (m, 5H), 1.19 (d, J=7.1 Hz, 3 H), 0.93 (t, J=8.8 Hz, 2 H), 0.89 (t, J=8.2 Hz,2 H), 0.02-0.01 (s, s, 18 H); Mass spec [M+Na]⁺ 992.

Fmoc-L-Ala-D-γ-Glu(O-TMSE)-L-Lys(N-TEOC)-D-Ala-D-Ala-OCH₃ (100 mg, 0.104mmol) is dissolved in 2 mL of 20% piperidine/DMF and stirred for 30 minat room temperature. Solvent is evaporated in vacuo, and the residue ispurified by flash chromatography (EtOAc, followed by 10% MeOH/CHCl₃) togive 60 mg (78%) of the desired product. R_(f)0.23 (10% MeOH/CHCl₃).

6.13. MurG Activity Assay Procedure

6.13.1. Protein Preparation

The wild type murG gene is cloned into the pET3a plasmid and transformedinto the high-stringency expression host BL21(DE3)pLysS (Novagen). Thelysogenized cells are grown at 37° C. in 2×YT media supplemented with 20μg/mL ampicillin and 34 μg/mL chloramphenicol to an O.D._(600 nm)=0.7;overexpression of the murG protein is achieved by induction for 1.25 hwith 1 mM IPTG. SDS/PAGE analysis shows production of a single new bandmigrating at ˜38,000 MW. Several hundred aliquots of the induced cellculture are prepared by centrifuging 1.0 mL samples at 5000 rpm for 10min at 4° C. The supernatant is removed, and the pellet frozen at −20°C. Frozen pellet stocks of non-transformed BL21(DE3)pLysS culture arealso prepared as a negative control. Protein quantitation using aprecipitated Lowry assay (Sigma) with a BSA reference on the entirepellet shows total protein concentration to be 11 and 17 μg/pellet forthe BL21(DE3)pLysS and overexpressed cell cultures, respectively.Immediately prior to reactions, pellets are thawed on ice andresuspended in 100 μL 1×R×n buffer.

6.13.2. Reaction Conditions

Biotinylated lipid substrate is aliquoted in autoclaved, sterile,deionized H₂O into 0.5 mL autoclaved Eppendorf tubes containing R×nbuffer (1×: 100 mM Tris-Cl pH 7.6, 1 mM MgCl₂). The ethanol is removedfrom an ethanol:water solution of ¹⁴C-UDP-GlcNAc (NEN Dupont) using anunheated SpeedVAC and then added to the substrate mixture (1.1×10⁵ DPM;r×n concentration of 9.4 μM). Finally, 5 μL iced crude cell lysatecontaining 0.5-1.0 μg protein are added to a total volume of 20 μL. Allreactions are performed at 24° C. Reactions are quenched by the additionof 10 μL 1% (w/v) SDS.

6.13.3. Transferase Activity Determination

A molar excess of biotin-binding TetraLink Tetrameric Avidin Resin(Promega) and deionized H₂O are added to each quenched reaction tube toa final volume of 350 μL. The suspension is incubated at roomtemperature for 10 min with frequent vortexing and transferred to anempty 1.5 mL microcolumn tube with a 30 μm frit (Bio-Rad). The resin iswashed (5×0.5 mL) using deionized H₂O. Washed resin is transferred using1.0 mL sterile, deionized H₂O to 10 mL Ecolite (ICN) and vortexed.Samples are counted immediately.

The results of various experiments are graphically depicted in FIG. 1.

6.14. Purification of Wild Type E. coli MurG

BL21(DE3)pLysS cells (Novagen) overexpressing wild type E. coli MurGfrom a pET3a vector (Novagen) are grown in 8L 2XYT medium supplementedwith 100 μg/mL ampicillin and 34 μg/mL chloramphenicol. When theOD_(600nm) reached 0.6, IPTG is added to a final concentration of 1 mM.The induced cell culture is grown for another 3.5 hours and then thecells are spun down in 500 mL batches at 5000 rpm (Beckman RC5Bcentrifuge) for 10 minutes and the supernatant is decanted. Each cellpellet is resuspended in 5 mL 25 mM MES (pH 6.0), 4 mM DTT and 3% TritonX-100, and the suspensions are combined for a total of 80 mL, and thenfrozen at −70° C. The suspension is thawed at 4° C., and to it is addedMgCl₂ to a final concentration of 5 mM and DNAse to a finalconcentration of 20 μg/mL. After shaking for 1 hour at 4° C., the debrisis spun down at 15,000 rpm for 35 minutes. The supernatant is decanted,diluted 6-fold with Buffer A (25 mM MES pH 6.0, 4 mM DTT), and appliedto a SP-Sepharose column (Pharmacia Biotech) equilibrated with Buffer A.After washing for 40 minutes with 40% Buffer B (20 mM Tris pH 8.0, 1MNaCl, 4 mM DTT)/Buffer A, the bound enzyme is eluted using a linear saltgradient starting with 40% Buffer B and ending with 100% Buffer B over120 minutes. The eluted enzyme is concentrated to 7 mg/mL and applied toa Superdex 200 HR 10/30 column (Pharmacia Biotech) at a flow rate of 0.5mL/min of TBSE buffer (100 mM NaCl, 20 mM Tris pH 8.0, 10 mM EDTA and 4mM DTT). The protein eluted as a single, symmetric peak at an estimatedmolecular weight of 72 kD. The purity of the enzyme is estimated to begreater than 98% from Coomassie Blue-stained SDS-polyacrylamide gel. Theyield of purified enzyme is approximately 1.3 mg/L of bacterial culture.The purified enzyme is stored at 4° C., and is stable for at least onemonth.

The following examples are best related to FIGS. 2-8 of thespecification.

6.15. Initial Rate Assays with Purified, Soluble Enzyme

The following solutions are prepared prior to the assays: 1) 177.3 μM[¹⁴C]-UDP-GlcNAc in H₂O (0.05 mCi/mL); 2) 1.5 mM UDP-GlcNAc in H₂O; 3)biotinylated Lipid I analog (1b) at 0.5 μg/μL; 4) 10×reaction buffercontaining 50 mM HEPES (pH 7.9) and 5 mM MgCl₂. The enzyme stock isprepared by diluting the purified enzyme with TBSE to a finalconcentration of 0.04 μg/μL in a 0.5 mL tube and storing at 4° C. fortwo days prior to running the assays.

Thirty reactions are prepared by individually mixing 2 μL of 10×reactionbuffer with an appropriate amount of biotinylated Lipid I analog (1b),radioactive UDP-GlcNAc, nonradioactive UDP-GlcNAc, and H₂O to a finalvolume of 18 μl. The final concentration for the Lipid I analog (1b) are7 μM, 10 μM, 15 μM, 30 μM, 100 μM, and for UDP-GlcNAc 11 μM, 15 μM, 20μM, 40 μM, 100 μM, 200 μM. Reactions are initiated by adding 2 μL of theenzyme stock and are run for 4 minutes at 24° C. Reactions are stoppedby adding 10 μl of 1% (w/v) SDS.

Radiolabeled product is separated from radiolabeled starting material byincubating a 3-fold molar excess of biotin-binding TetraLink TetramericAvidin Resin (Promega) to each tube. Deionized H₂O is added to each tubeto a final volume of approximately 250 μL and the suspension istransferred to a 1.0 μM pore size 96-well filter plate fitted to avacuum-line fitted MultiScreen Assay System (Millipore). The resin iswashed 15 times with 0.2 mL deionized H₂O. Washed resin is transferredto a scintillation vial containing 10 mL Ecolite and vortexed. Samplesare counted immediately on a Beckman LS5000 scintillation counter.

6.16. IC₅₀ Measurements

The IC₅₀ assays are performed the same way as the initial rate assaysexcept that the Lipid I analog (1) and UDP-GlcNAc concentrations arefixed at 18 μM and 34.3 μM, respectively. Each set of assays is carriedout at five or six different concentrations of one of the inhibitorycompounds. The IC₅₀ is taken as the concentration at which the reactionrate (counts incorporated in a given time) decreased by 50%.

6.17. General Methods

All amino acids are purchased from BAChem. Unless otherwise stated, allchemicals are purchased from Aldrich or Sigma and used without furtherpurification. Dichloromethane, toluene, benzene, pyridine,diisopropylethylamine and triethylamine are distilled from calciumhydride under dry argon. Diethyl ether and tetrahydrofuran are distilledfrom potassium benzophenone under dry argon. DMF, ethyl acetate andmethanol are dried over activated molecular sieves.

Analytical thin layer chromatography (TLC) is performed on silica gel 60F₂₅₄ plates (0.25 mm thickness) protected with a fluorescent indicator.The developed plates are examined under short wave UV light and stainedwith anisaldehyde or Mo (Vaughn) stain. Flash chromatography isperformed using silica gel 60 (230-400 mesh) from EM Science.

NMR spectra are recorded on a JEOL GSX-270 NMR spectrometer or a VarianInova 500/VNMR spectrometer. Chemical shifts (δ) are reported in partsper million (ppm) downfield from tetramethylsilane. Coupling constants(J) are reported in Hertz (Hz). Multiplicities are abbreviated asfollows: singlet (s), doublet (d), triplet (t), quartet (q), multiplet(m), double of doublets (dd), apparent triplet (apt), broad singlet(bs), pentet (p), and octet (o).

High-resolution mass spectra (FAB) are obtained by Dr. Ron New at theUniversity of California at Riverside Department of Chemistry MassSpectrometry Facility. Low-resolution mass spectra (ESI) are obtained byDr. Dorothy Little at the Princeton University Department of Chemistry.

6.17.1. Compound 3

To a solution of compound 2 (482 mg, 1.02 mmol; see, FIG. 7) and4-dimethylaminopyridine (10 mg, 0.08 mmol) in 8 mL of THF is addedtrichloroethanol (0.23 mL, 2.40 mmol) followed by1,3-dicyclohexylcarbodiimide (248 mg, 1.20 mmol). After stirring at roomtemperature for 4 hours, the reaction solution is filtered throughcotton plug and the precipitate is rinsed with EtOAc. The filtrate isconcentrated and purified by flash chromatography (15% EtOAc/CH₂Cl₂) togive 453 mg (80%) of 3 as a white powder. R_(f)0.39 (15% EtOAc/CH₂Cl₂);¹H NMR (CDCl₃, 500 MHz) δ7.43-7.25 (m, 10 H), 7.07 (d, J=6.0 Hz, 1 H),5.59 (s, 1 H), 5.34 (d, J=3.2 Hz, 1 H), 4.98 (d, J=11.9 Hz, 1 H),4.68(d, J=12.0 Hz, 1 H), 4.66 (q, J=7.0 Hz, 1 H), 4.60 (d, J=11.9 Hz, 1H), 4.51 (d, J=12.0 Hz, 1 H), 4.21 (dd, J=10.5, 4.8 Hz, 1 H), 4.00 (m, 1H), 3.85 (m, 2 H), 3.75 (m, 2 H), 2.04 (s, 3 H), 1.50 (d, J=7.0 Hz, 3H); ¹³C NMR (CDCl₃, 500 MHz) δ173.8, 170.9, 137.5, 137.4, 129.3, 128.6,128.5, 128.1, 128.0, 126.1, 101.6, 97.5, 94.6, 83.4, 75.2, 75.1, 74.3,70.5, 69.2, 63.1, 54.2, 23.4, 18.9; HRMS(FAB) calcd for C₂₇H₃₁NO₈Cl₃[M+H⁺]: 602.1115, found: 602.1130.

6.17.2. Compound 4

To a solution of compound 3 (360 mg, 0.60 mmol) in 30 mL of EtOAc isadded 500 mg of 20% Pd-C. The reaction vessel is filled with hydrogen.After stirring at room temperature for 30 minutes, the suspension isfiltered and the catalyst is rinsed with methanol. The filtrate isconcentrated to give a clear oil which is used in the next reactionwithout further purification.

To a solution of this clear oil in 6 mL of DMF is added benzylaldehydedimethyl acetal (0.9 mL, 6.0 mmol) followed by p-toluenesulfonic acid(11.4 mg, 0.06 mmol). The reaction is stirred at room temperature for 10hours and neutralized with saturated NaHCO₃. Then the mixture isextracted with CH₂Cl₂ (3×20 mL). The CH₂Cl₂ layers are combined, driedover anhydrous sodium sulfate, filtered, concentrated, and purified byflash chromatography (90% EtOAc/petroleum ether) to give 248 mg (81%) of4 as a mixture of α, β anomers (α:β=4:1). R_(f) (α anomer) 0.33, R_(f)(β anomer) 0.28 (90% EtOAc/petroleum ether); α anomer ¹H NMR (CDCl₃, 270MHz) δ7.50-7.35 (m, 5 H), 5.66 (bs, 1 H), 5.58 (s, 1 H), 5.02 (d, J=12.0Hz, 1 H), 4.95 (m, 1 H), 4.67 (m, 1 H), 4.58 (d, J=12.0 Hz, 1 H), 4.27(dd, J=10.0, 5.0 Hz, 1 H), 4.05 (m, 1 H), 2.06 (s, 3 H), 1.52 (d, J=7.0Hz, 3 H); ¹³C NMR (CDCl₃, 270 MHz) δ174.2, 171.9, 137.6, 129.2, 128.5,126.2, 101.5, 94.7, 91.4, 83.5, 75.5, 75.0, 74.6, 69.2, 62.9, 54.9,23.4, 18.9; HRMS(FAB) calcd for C₂₀H₂₅NO₈Cl₃ [M+H⁺]; 512.0646, found:512.0653.

6.17.3. Compound 5

Compound 4 (202 mg, 0.40 mmol) and 1H-tetrazole are premixed andco-evaporated with toluene and dissolved in 10 mL of CH₂Cl₂. Thereaction solution is cooled to −30° C. and dibenzylN,N-diisopropylphosphamide (0.27 mL, 0.79 mmol) is added. The reactionis warmed up to room temperature in 30 minutes and stirred for anotherhour. Then the reaction is cooled to −40° C. and m-CPBA (560 mg, 2 mmol)is added. After stirring for 30 minutes at 0° C. and another 30 minutesat room temperature, the reaction is diluted with 20 mL of CH₂Cl₂,extracted with 10% aqueous Na₂SO₃ (2×20 mL), saturated NaHCO₃ (2×20 mL),and water (2×20 mL). The CH₂Cl₂ layer is dried over anhydrous sodiumsulfate, filtered, concentrated, and purified by flash chromatograghy(65% EtOAc/petroleum ether) to give 200 mg (70%) of 5 as a white solid.R_(f)0.24 (70% EtOAc/petroleum ether); ¹H NMR (CDCl₃, 500 MHz)δ7.44-7.33 (m, 15 H), 7.20 (d, J=6.0 Hz, 1 H), 6.10 (m, 1 H), 5.56 (s, 1H), 5.07 (m, 4 H), 5.02 (d, J=12.0 Hz, 1 H), 4.64 (q, J=7.0 Hz, 2 H),4.59 (d, J=12.0 Hz, 1 H), 4.09 (m, 1 H), 4.03 (m, 1 H), 3.95 (m, 1 H),3.83-3.68 (m, 3 H), 1.86 (s, 3 H), 1.48 (d, J=7.0 Hz, 3 H); ¹³C NMR(CDCl₃, 500 MHz) δ173.8, 171.2, 137.1, 129.4, 128.8, 128.5, 128.2,128.0, 126.1, 101.7, 96.2, 96.1, 82.6, 75.3, 74.3, 74.2, 69.7, 68.6,64.6, 54.2, 54.1, 23.0, 18.8; HRMS(FAB) calcd for C₃₄H₃₇NO₁₁Cl₃PNa[M+Na⁺]: 794.1068, found 794.1095.

6.17.4. Compound 6

To a solution of compound 5 (58 mg, 0.075 mmol) in 5 mL of 90% AcOH/H₂Ois added zinc dust (30 mg). The reaction is stirred vigorously at roomtemperature for 1 hour. The suspension is filtered and the precipitateis rinsed with methanol. The filtrate is concentrated and purified byflash chromatography (10% MeOH/CHCl₃/0.1% AcOH) to give 44 mg (91%) of 6as a white solid. R_(f)0.19 (5% MeOH/CHCl₃, 0.1% AcOH); ¹H NMR (CD₃OD,500 MHz) δ7.44-7.25 (m, 15 H), 6.11 (m, 1 H), 5.55 (s, 1 H), 5.02 (m, 4H), 4.33 (q, J=7.0 Hz, 1 H), 3.96 (m, 1 H), 3.77 (m, 1 H), 3.7-3.66 (m,4 H), 1.94 (s, 3 H), 1.32 (d, J=7.0 Hz, 3 H); ¹³C NMR (CD₃OD, 500 MHz)δ181.2, 174.2, 139.0, 137.1, 130.0, 129.9, 129.3, 129.2, 127.3, 102.8,97.4, 83.2, 78.3, 75.0, 71.2, 69.2, 66.4, 56.2, 56.1, 22.8, 19.7;HRMS(FAB) calcd for C₃₂H₃₆NO₁₁PNa [M+Na⁺]: 664.1924, found 664.1938.

6.17.5. Compound 7

6.17.5.1. Fmoc-L-Lys(N-TEOC)-OH

To a solution of Fmoc-L-Lys(N-BOC)-OH (607 mg, 1.30 mmol) in 10 mL ofCH₂Cl₂ is added 10 mL of trifluoroacetic acid. The mixture is stirredfor 20 minutes at room temperature and concentrated. The residue isdissolved in 10 mL of DMF. Diisopropylethylamine (1.1 mL, 6.48 mmol) isadded. 2-(Trimethylsilyl)ethyl p-nitrophenyl carbonate (440 mg, 1.55mmol) is dissolved in 3 mL of DMF and transferred into the reactionsolution. After stirring for 2 hours at room temperature, solvent isremoved under vacuum. The residue is purified by flash chromatography(eluting first with EtOAc then with 10% MeOH/CHCl₃/0.1% AcOH) to give635 mg (95%) of the desired product as a white solid. R_(f)0.54 (10%MeOH/CHCl₃).

6.17.5.2. Z-D-Glu(OH)-OTMSE

To a solution of Z-D-Glu(O-bzl)-OH (1.1 g, 3.0 mmol) and DMAP (37 mg,0.3 mmol) in 30 ML of EtOAc is added DCC (0.7 g, 3.6 (mmol) and2-Trimethylsilyl)ethanol (0.5 mL, 3.6 mmol). After stirring for 20minutes at room temperature, the reaction is filtered. The filtrate isconcentrated and purified by flash chromatography (15% EtOAc/petroleumether) to give 1.3 g (91%) of Z-D-Glu(O-bzl)-OTMSE as a white solid.R_(f)0.30 (15% EtOAc/petroleum ether).

To a solution of Z-D-Glu(O-bzl)-OTMSE (1.2 g, 2.6 mmol) in 30 mL of MeOHis added 900 mg of 20% Pd—C. After stirring for 10 minutes at roomtemperature, the suspension is filtered. The filtrate is concentratedand dissolved in 20 mL of H₂O/dioxane (1:1). To the solution is addedNaHCO₃ (0.44, 5.2 mmol). A solution of Cbz-succinimide (0.8 g, 3.1 mmol)in 5 mL of dioxane is added to the reaction over 30 minutes. Then 1 mLof AcOH is added. Solvent is removed under vacuum. The residue ispurified by flash chromatography (eluting first with 10% EtOAc/CH₂Cl₂then with 10% MeOH/CHCl₃/0.1% AcOH) to give 0.9 g (87%) ofZ-D-Glu(OH)-OTMSE as a white solid. R_(f)0.49 (10% MeOH/CHC₃); ¹H NMR(CD₃OD, 500 MHz) δ7.24-7.15 (m, 5 H), 4.96 (d, J=3.0 Hz, 1 H), 4.10 (m,4H), 2.28 (t, J=7.6 Hz, 2 H), 2.02 (m, 1 H), 1.80 (m, 1 H), 0.87 (t,J=8.6 Hz, 2 H), −0.08 (s, 9 H); ¹³C NMR (CD₃OD, 500 MHz, δ176.3, 173.9,158.6, 138.2, 129.5, 129.1, 128.9, 67.7, 64.7, 55.0, 31.2, 27.8, 18.2,−1.3;

Peptide 7 is synthesized by standard HOBt/HBTU method with Fmocprotected amino acids. R_(f)0.29 (10% MeOH/CHCl₃); ¹H NMR (DMSO, 500MHz) δ8.15 (d, J=5.0 Hz, 1 H), 8.14 (d, J=5.0 Hz, 1 H), 8.10 (d, J=8.0Hz, 1 H), 8.02 (d, J=5.0 Hz, 1 H), 6.92 (t, J=5.0 Hz, 1 H), 4.30 (m, 1H), 4.19 (m, 2 H), 4.17-4.07 (m, 5 H), 4.00 (t, J=8.5 Hz, 2 H), 3.31 (q,J=8.5 Hz, 1 H), 2.92 (m, 2 H), 2.18 (m, 2 H), 1.95 (m, 1 H) 1.80 (m, 1H), 1.57 (m, 1 H), 1.48 (m, 1 H), 1.36 (m, 2 H), 1.29 (d, J=8.5 Hz, 3H), 1.25 (m, 2 H), 1.20 (d, J=8.5 Hz, 3 H), 1.13 (d, J=8.5 Hz, 3 H),0.92 (m, 6 H), 0.02-0.00 (3s, 27 H); ¹³C NMR (DMSO, 500 MHz) δ175.8,172.3, 172.0, 171.8, 171.5, 171.4, 156.2, 62.6, 62.4, 61.2, 52.9, 51.3,50.1, 47.7, 47.6, 31.4, 31.2, 29.2, 27.2, 22.6, 21.4, 18.0, 17.4, 16.9,16.8, 16.7, −1.4, −1.5, −1.6; HRMS(FAB) calcd for C₃₆H₇₂N₆O₁₀Si₃Na[M+Na⁺]: 855.4515, found: 855.4564.

6.17.6 Compound 8

To a solution of compound 6 (85 mg, 0.13 mmol) andNH₂-L-Ala-□-D-Glu(O-TMSE)-L-Lys(N-TEOC)-D-Ala-D-Ala-OTMSE (7) (153 mg,0.18 mmol) in 1.5 mL of DMF is added diisopropylethylamine (116 μL, 0.66mmol) followed by HOBt (27 mg, 0.20 mmol) and PyBOP (104 mg, 0.20 mmol).After stirring for 30 minutes at room temperature, the solution isdiluted in 10 mL of EtOAc and washed with 0.01 N aqueous HCl (3×10 mL).The organic layer is concentrated, dried over anhydrous sodium sulfate,and purified by flash chromatograghy (5% MeOH/CHCl₃) to give 168 mg(87%) of 8 as a white solid. R_(f)0.24 (5% MeOH/CHCl₃); ¹H NMR (CD₃OD,500 MHz) δ7.52-7.37 (m, 15 H), 5.88 (m, 1 H), 5.65 (s, 1 H), 5.13 (m, 4H), 4.41 (m, 2 H), 4.35 (m, 3 H), 4.17 (m, 8 H), 4.06 (dd, J=9.5, 3.5Hz, 1 H), 3.84 (m, 3 H), 3.77 (m, 1 H), 3.10 (m, 2 H), 2.29 (t, J=14.5Hz, 2 H), 2.19 (m, 1 H), 1.90 (m, 1 H), 1.88 (s, 3 H), 1.77 (m, 1 H),1.67 (m, 1 H), 1.51 (m, 2 H), 1.43-1.35 (m, 14 H), 1.01-0.97 (m, 6 H),0.06-0.04 (3s, 27 H); ¹³C NMR (CDCl₃, 500 MHz) δ173.9, 172.8, 172.4,171.8, 171.3, 157.1, 137.1, 135.5, 135.4, 129.2, 129.0, 128.9, 128.7,128.4, 128.1, 126.1, 101.6, 97.1, 82.5, 81.0, 78.2, 76.7, 70.0, 69.6,68.4, 64.8, 64.1, 63.8, 63.0, 53.9, 53.3, 51.4, 50.0, 49.1, 48.4, 40.4,31.6, 31.5, 29.6, 27.9, 23.1, 22.7, 19.6, 18.0, 17.9, 17.8, 17.5, 17.4,−1.3, −1.4, −1.5; HRMS(FAB) calcd for C₆₈H₁₀₆N₇O₂₀PSi₃Na [M+Na⁺]:1478.6436, found: 1478.6417.

6.17.7 Compound 9

To a solution of compound 8 (87 mg, 0.06 mmol) in 5 mL of MeOH is added20 mg of 20% Pd—C. The reaction vessel is filled with hydrogen andstirred at room temperature. 1 ml of pyridine is added after 30 minutes.The solution is diluted with 15 mL of MeOH and stirred for 30 minutes.The catalyst is filtered off. The filtrate is concentrated to giveproduct 9a which is used in the next reaction without furtherpurification. R_(f)0.28 (CHCl₃:MeOH:H₂O=3:2:0.5).

Citronellol phosphate (25 mg, 0.11 mmol) [Ref: Warren, C. D., Jeanloz,R. W., Biochem, 14, 412-419, 1975] is coevaporated with toluene (3×1 mL)and dissolved in 2 mL of CH₂Cl₂. Diisopropylethylamine (92 μL, 0.53mmol) is added. The solution is cooled to −20° C. anddiphenylphosphorochloridate (26 μL, 0.13 mmol) is added. The reaction isallowed to warm up to room temperature in 10 minutes and stirred at roomtemperature. After 1 hour, methanol (1 mL) is added and the reaction isstirred for another hour at room temperature. Solvent is removed undervacuum. The residue is coevaporated with toluene (3×1 mL) and dissolvedin 0.5 mL of CH₂Cl₂.

Compound 9a (58 mg, 0.04 mmol) is coevaporated with toluene (3×1 mL) anddissolved in 1 mL of CH₂Cl₂. 0.4 mL of the citronelloldiphenylpyrophosphate solution is added to the reaction followed bypyridine (20 μL, 0.24 mmol). The reaction is stirred at room temperaturefor 18 hours. Solvent is removed under vacuum and the residue is loadedto a C18 reverse phase column (8 mm×80 mm, particle size 40 μm, poresize 60 A, from J. T. Baker) and eluted with CH₃CN/0.1% NH₄HCO₃ aqueoussolution (0, 5%, 10%, 15%, 20%, 25%, 30%, 35% of 10 mL each). Thefractions containing desired product are combined and concentrated togive 34 mg (68%) of 9 as a white powder. R_(f)0.21 (CHCl₃:MeOH:H₂O=4.5:1.5: 0.2). This product is used in the next reaction without furtherpurification. ESI-MS calcd for C₅₇H₁₀₉N₇O₂₃P₂Si₃Na [M+Na⁺]: 1429, found:1429.

6.17.8 Compound 1a

To a solution of compound 9 (43 mg, 0.023 mmol) in 0.7 mL of DMF isadded tetrabutylammonium fluoride (1 M in THF, 0.7 mL). The reaction isstirred at room temperature for 24 hours. Solvent is removed undervacuum. The residue is loaded to a C18 reverse phase column (8 mm×80 mm,particle size 40 μm, pore size 60 A, from J. T. Baker), and eluted withCH₃CN/0.1% NH₄HCO₃ aqueous solution (0, 5%, 10%, 15%, 20%, 25%, 30% of10 mL each. The fractions containing the desired product are combinedand concentrated. The crude product is further purified on adiethylaminoethyl cellulose column (14 mm×80 mm, from Whatman Labsales,Inc.), eluted with 250 mM NH₄HCO₃, to give 24 mg of 1a (93%) as a whitepowder after lyophilization. R_(f)0.18 (CHCl₃:MeOH:H₂O=3:3:1); ¹H NMR(CD₃OD, 500 MHz) δ5.58 (m, 1 H), 5.11 (t, J=6.5 Hz, 1 H), 4.50-3.56 (m,12 H), 2.94 (m, 2 H), 2.34 (m, 2H), 2.10 (s, 3 H), 2.00 (m, 1 H), 1.98(m, 2 H), 1.92 (m, 1 H), 1.74 (m, 2 H), 1.67 (s, 3 H), 1.62 (m, 1 H),1.60 (s, 3 H), 1.50-1.39 (m, 12 H), 1.23 (m, 2 H), 0.93 (d, J=6.5 Hz, 3H); ¹³C NMR (D₂O, 500 MHz) δ178.2, 177.9, 176.7, 176.6, 176.5, 176.4,176.3, 165.3, 135.5, 127.6, 97.0, 82.2, 80.3, 75.4, 74.1, 72.0, 71.9,71.8, 70.4, 67.6, 62.7, 56.6, 55.8, 52.3, 51.9, 51.2, 41.5, 38.8, 34.0,32.7, 31.0, 30.0, 28.6, 27.2, 27.1, 24.6, 24.4, 21.0, 19.2, 19.1, 18.8;ESI-MS calcd for C₄₁H₇₄O₂₁N₇P₂ [M+H³⁰ 9 : 1062, found: 1062.

6.17.9 Compound 1b

To a solution of compound 1a (25 mg, 0.022 mmol) in 1.5 mL ofH₂O/dioxane(1:1) is added NaHCO₃ (23 mg, 0.4 mmol) followed by6-((biotinoyl)amino)hexanoic acid succinimide ester (12 mg, 0.027 mmol).The reaction is stirred at room temperature for 2 hours. Solvent isremoved under vacuum. The residue is loaded on a diethylaminoethylcellulose column (14 mm×80 mm, from Whatman Labsales, Inc.), eluted with250 mM NH₄HCO₃ to give 16 mg (80%) of 1b as a white powder afterlyophilization. R_(f)0.40 (CHCl₃:MeOH:H₂O=3:3:1); ¹H NMR (CD₃OD, 500MHz) δ5.49 (dd, J=3.0, 7.3 Hz, 1 H), 5.11 (t, J=7.2 Hz, 1 H), 4.50 (dd,J=4.8 7.8 Hz, 1 H), 4.37 (m, 2 H), 4.31 (dd, J=4.3, 7.8 Hz, 1 H), 4.29(m, 1 H), 4.24 (m, 3 H) 4.16 (d, J=10.4 Hz, 1 H), 4.02 (m, 2 H), 3.99(m, 1 H), 3.90 (d, J=11.0 Hz, 1 H), 3.74 (m, 1 H), 3.70 (m, 1 H), 3.49(dd, J=9.5, 9.5 Hz, 1 H), 3.21 (m, 1 H), 3.17 (m, 4 H), 2.94 (dd, J=4.8,12.8 Hz, 1 H), 2.71 (d, J=12.8 Hz, 1 H), 2.31 (m, 1 H), 2.28 (m, 2 H),2.25 (m, 1 H), 2.20 (m, 4 H), 2.02 (s, 3 H), 2.00 (m, 2 H), 1.86 (m, 2H), 1.82 (m, 1 H), 1.73 (m, 4 H), 1.67 (s, 3 H), 1.63 (m, 5 H), 1.61 (s,3 H), 1.52 (m, 4 H), 1.45 (m, 2 H), 1.44 (d, J=7.3 Hz, 3 H), 1.43 (d,J=6.2 Hz, 3 H), 1.41 (m, 2 H), 1.38 (d, J=7.3 Hz, 3 H), 1.37 (d, J=7.2Hz, 3 H), 1.35 (m, 2 H), 1.17 (m, 1 H), 0.93 (d, J=6.7 Hz, 3 H); ¹³C NMR(CD₃OD, 500 MHz) δ177.2, 176.5, 176.2, 176.1, 176.0, 175.6, 174.7,174.6, 174.5, 174.2, 166.3, 132.1, 126.2, 96.4, 81.3, 78.8, 75.2, 71.0,65.7, 63.6, 63.0, 61.8, 57.2, 55.7, 55.0, 54.2, 50.9, 50.7, 50.4, 41.2,40.4, 40.2, 39.1, 39.0, 38.6, 37.2, 37.0, 33.0, 32.5, 30.6, 30.3, 30.2,30.0, 29.6, 27.7, 27.1, 26.9, 26.7, 26.1, 24.5, 23.5, 20.0, 19.5, 18.4,18.3, 18.0, 17.9; HRMS(FAB) calcd for C₅₇H₉₅N₁₀O₂₄P₂SNa [M−3H⁺+2Na⁺]:1443.5512, found: 1443.5494.

6.17.10 Compound 10

Compound 10 is made following the same scheme as 1a except that in stepe, intermediate 6 is coupled to dipeptideCH₃NH-D-γ-Glu(O-TMSE)-L-Ala-NH₂ instead of to 7. R_(f)0.41(CHCl₃:MeOH:H₂O=3:3:1); ¹H NMR (CD₃OD, 500 MHz) δ5.49 (dd, J=3.0, 7.0Hz, 1 H), 5.11 (t, J=6.6 Hz, 1 H), 4.33 (q, J=7.0 Hz, 1 H), 4.27 (q,J=7.0 Hz, 1 H), 4.24 (dd, J=3.8, 7.6 Hz, 1 H), 4.16 (m, 1 H), 4.04 (m, 2H), 4.00 (m, 1 H), 3.90 (dd, J=1.8, 11.8 Hz, 1 H), 3.75 (dd, J=9.6, 9.6Hz, 1 H), 3.70 (dd, J=5.7, 11.8 Hz, 1 H), 3.48 (dd, J=9.6, 9.6 Hz, 1 H),2.64 (s, 3 H), 2.18 (m, 2 H), 2.16 (m, 1 H), 2.02 (s, 3 H), 1.98 (m, 2H), 1.92 (m, 1 H), 1.72 (m, 1 H), 1.67 (s, 3 H), 1.62 (m, 1 H), 1.61 (s,3 H), 1.47 (m, 1 H), 1.43 (d, d, J=7.0 Hz, 6 H), 1.37 (m, 1 H), 1.18 (m,1 H), 0.94 (d, J=6.6 Hz, 3 H); ¹³C NMR (CD₃OD, 500 MHz) δ177.2, 176.1,176.0, 174.4, 174.2, 132.0, 126.1, .96.3, 81.1, 78.9, 75.2, 70.8, 65.7,63.0, 55.1, 54.9, 51.0, 39.1, 38.6, 33.3, 30.6, 30.1, 26.7, 26.5, 26.1,23.4, 19.9, 19.5, 18.2, 17.9; HRMS(FAB) calcd for C₃₀h₅₃N₄O₁₇P₂ [M−H⁺]:803.2881, found: 803.2861.

6.17.11 Compound 11a

Compound 11a is made following the same scheme as 1a except that in stepe, compound 6 is coupled to TEOC-NHCH₂CH₂NH₂ instead of to 7. The silylprotecting group is cleaved using TBAF, the same as in making 1a.R_(f)0.20 (CHCl₃:MeOH:H₂O=3:2:0.5); ¹H NMR (CD₃OD, 500 MHz) δ5.58 (bs, 1H), 5.11 (t, J=7.0 Hz, 1 H), 4.30 (q, J=6.7 Hz, 1 H), 4.21 (m, 1 H),4.04 (m, 3 H), 3.72 (m, 1 H), 3.78 (m, 1 H), 3.73 (m, 1 H), 3.64 (m, 1H), 3.50 (dd, J=9.4, 9.4 Hz, 1 H), 3.40 (m, 1 H), 3.13 (m, 2 H), 2.03(s, 3 H), 2.00 (m, 2 H), 1.73 (m, 1 H), 1.67 (s, 3H), 1.63 (m, 1 H),1.61 (s, 3 H), 1.46 (m, 1 H), 1.39 (m, 1 H), 1.38 (d, J=6.7 Hz, 3), 1.18(m, 1 H), 0.94 (d, J=6.7 Hz, 3 H); ¹³C NMR (CD₃OD, 500 MHz) δ176.2,173.6, 131.2 125.2, 95.6, 80.7, 78.1, 74.3, 70.3, 64.9, 62.0, 54.2,39.7, 38.2, 37.8, 37.5, 29.8, 25.8, 25.2, 22.5, 19.1, 18.6, 17.0;HRMS(FAB) calcd for C₂₃H₄₃N₃O₁₃P₂Na [M−2H⁺+Na⁺]: 654.2169, found654.2199.

6.17.12 Compound 11b

Compound 11a (4 mg, 0.006 mmol) and 4-nitrophenyl acetate (1.2 mg, 0.007mmol) is dissolved in 0.4 mL of DMF. Large amount of KHCO₃ is added toincrease PH. Equal amount of 4-nitrophenyl acetate is added every 12hours. After 3 days, the reaction is completed. The solvent is removedis removed and the residue is loaded to a C18 reverse phase column (8mm×80 mm, particle size 40 μm, pore size 60 A, from J. T. Baker) andeluted with CH₃CN/0.1% NH₄HCO₃ aqueous solution (0, 5%, 10%, 15%, 20%,25%, 30%, 35% of 10 mL each). The fractions containing desired productare combined and concentrated to give 3 mg (71%) of 11b as a whitepowder. R_(f)0.26 (CHCl₃:MeOH:H₂O=3:2:0.5); ¹H NMR (CD₃OD, 500 MHz)δ5.50 (bs, 1 H), 5.12 (t, J=7.0 Hz, 1 H), 4.22 (q, J=7.0 Hz, 1 H), 4.04(m, 2 H), 4.00 (m, 1 H), 3.89 (d, J=12.2 Hz, 1 H), 3.72 (m, 2 H), 3.46(dd, J=9.5, 9.5 Hz, 1 H), 3.36 (m, 2 H), 3.28 (m, 2 H), 2.04 (s, 3 H),2.02 (m, 2 H), 1.98 (s, 3 H), 1.73 (m, 1 H), 1.68 (s, 3 H), 1.63 (m, 1H), 1.62 (s, 3 H), 1.46 (m, 1 H), 1.40 (d, J=7.0 Hz, 3 H), 1.38 (m, 1H), 1.18 (m, 1 H), 0.94 (d, J=6.7 Hz, 3 H);. ¹³C NMR (CD₃OD, 500 MHz)δ176.4, 174.5, 173.8, 132.0, 126.1, 96.4, 81.9, 79.2, 75.1, 71.0, 65.6,62.9, 55.0, 40.2, 40.1, 39.0, 38.6, 30.6, 26.7, 26.0, 23.4, 22.8, 19.9,19.5, 17.9; HRMS(FAB) calcd for C₂₅H₄₆N₃O₁₄P₂ [M−N⁺]: 674.2455, found674.2488.

6.17.13 Compound 11c

Compound 11c is made from 11a and 6-((biotinoyl)amino)hexanoic acidsuccinimide ester using the same chemistry described in step h (schemeII). R_(f)0.30 (CHCl₃:MeOH:H₂O=3:2:0.5); ¹H NMR (CD₃D, 500 MHz) δ5.49(dd, J=2.7, 7.0 Hz, 1 H), 5.12 (t, J=7.2 Hz, 1 H), 4.50 (dd, J=5.0, 7.5Hz, 1 H), 4.32 (dd, J=4.4, 7.5 Hz, 1 H), 4.20 (q, J=6.7 Hz, 1 H), 4.16(m, 1 H), 4.03 (m, 2 H), 3.98 (m, 1 H), 3.90 (d, J=12.0 Hz, 1 H), 3.71(m, 1 H), 3.70 (m, 1 H), 3.45 (dd, J=9.4, 9.4 Hz, 1 H), 3.23 (m, 1 H),3.18 (m, 6 H), 2.94 (dd, J=5.0, 12.8 Hz, 1 H), 2.72 (d, J=12.8 Hz, 1 H),2.24 (t, J=7.6 Hz, 2 H), 2.21 (t, J=7.6 Hz, 2 H), 2.03 (s, 3 H), 2.00(m, 2 H), 1.73 (m, 3 H), 1.68 (s, 3 H), 1.64 (m, 6 H), 1.62 (s, 3 H),1.53 (m, 2 H), 1.45 (m, 3 H), 1.40 (d, J=6.7 Hz, 3 H), 1.36 (m, 3 H),1.18 (m, 1 H), 0.94 (d, J=6.7 Hz, 3 H); ¹³C NMR (CD₃OD, 500 MHz) δ176.5,176.4, 176.1, 174.4, 166.3, 132.0, 126.1, 96.5, 81.9, 79.2, 75.2, 70.9,65.7, 63.5, 62.9, 61.8, 57.1, 55.0, 41.2, 40.4, 40.1, 39.1, 39.0, 38.6,37.2, 37.0, 30.6, 30.3, 30.0, 29.6, 27.8, 27.1, 26.8, 26.7, 26.1, 23.4,20.0, 19.6, 18.0; HRMS(FAB) calcd for C₃₉H₆₉N₆O₁₆P₂S [M−H⁺]: 971.3966,found: 971.3948.

6.17.14 Compound 12a

The intermediate from hydrogenation of compound 8 is deprotected withTBAF using the same method for making 1a. R_(f)0.16(CHCl₃:MeOH:H₂O=3:4:1.5); ¹H NMR (CD₃OD, 500 MHz) δ5.34 (dd, J=3.0, 7.0Hz, 1 H), 4.24 (m, 3 H), 4.17 (dd, J=6.7, 6.7 Hz, 1 H), 4.08 (dd, J=4.6,8.5 Hz, 1 H), 4.03 (q, J=7.0 Hz, 1 H), 3.93 (m, 1 H), 3.80 (m, 1 H),3.75 (m, 1 H), 3.59 (dd, J=5.5, 11.6 Hz, 1 H), 3.56 (m, 1 H), 3.38 (dd,J=9.7, 9.7 Hz, 1 H), 2.82 (t, J=7.3 Hz, 2 H), 2.22 (m, 2 H), 2.15 (m, 1H), 1.86 (s, 3 H), 1.70 (m, 4 H), 1.58 (m, 2 H), 1.40 (m, 1 H), 1.31 (m,6 H), 1.25 (m, 6 H); ¹³C NMR (CD₃OD, 500 MHz) δ179.4, 178.8, 178.0,176.2, 175.9, 174.7, 174.0, 173.8, 95.3, 81.2, 78.7, 74.9, 71.2, 62.8,55.5, 55.3, 55.0, 51.9, 51.1, 50.8, 40.5, 33.1, 32.5, 30.4, 28.4, 23.7,23.4, 19.8, 19.4, 18.4, 18.0; HRMS(FAB) calcd for C₃₁H₅₃N₇O₁₈P [M−H⁺]:842.3185, found: 842.3212.

6.17.15 Compound 12b

Compound 12b is made from 12a and 6-((biotinoyl)amino)hexanoic acidsuccinimide ester using the same chemistry described in step h (schemeII). R_(f)0.27 (CHCl₃:MeOH:H₂O=3:4:1.5); ¹H NMR (CD₃OD, 500 MHz) δ5.45(dd, J=7.0, 3.0 Hz, 1 H), 4.51 (dd, J=5.0, 7.5 Hz, 1 H), 4.39 (m, 2 H),4.32 (m, 2 H), 4.26 (m, 3 H), 4.12 (m, 1 H), 3.91 (m, 1 H), 3.86 (d,J=11.6 Hz, 1 H), 3.73 (dd, J=5.5, 11.6 Hz, 1 H), 3.69 (m, 1 H), 3.53 (m,1 H), 3.22 (m, 1 H), 3.17 (m, 4 H), 2.94 (dd, J=5.0, 12.8 Hz, 1 H), 2.72(d, J=12.8 Hz, 1 H), 2.30 (m, 4 H), 2.21 (m, 4 H), 1.99 (s, 3 H), 1.89(m, 1 H), 1.82 (m, 1 H), 1.74 (m, 2 H), 1.63 (m, 4 H), 1.53 (m, 4 H),1.46 (m, 2 H), 1.44 (m, 6 H), 1.39 (m, 6 H), 1.35 (m, 4 H); ¹³C NMR(CD₃OD, 500 MHz) δ177.5, 177.4, 177.3, 176.5, 176.3, 174.8, 174.7,174.6, 174.3, 174.2, 166.0, 94.3, 80.6, 78.6, 73.2, 68.9, 62.8, 61.1,60.9, 56.1, 55.0, 54.3, 54.2, 51.6, 50.4, 50.1, 40.4, 39.8, 39.6, 36.4,36.2, 32.5, 31.4, 28.8, 28.7, 28.6, 28.5, 28.4, 26.2, 25.9, 25.8, 23.2,22.7; HRMS(FAB) calcd for C₄₇H₇₈N₁₀O₂₁P₂S [M−H⁺]: 1181.4801, found:1181.4769.

6.17.16 Compound 13a

To a solution of compound 6 (12 mg, 0.019 mmol) in 1 mL of methanol isadded 10 mg of pearlman's catalyst. The reaction vessel is filled withhydrogen. After stirring at room temperature for 30 min, a few drops ofpyridine is added. The suspension is filtered after stirring for another30 min. The filtration is concentrated to give a yellow oil which ispurified on a diethylaminoethyl cellulose column (14 mm×80 mm, fromWhatman Labsales, Inc.), eluted with 1M NH₄HCO₃, to give 7 mg (90%) of13a as a white powder. R_(f)0.29 (CHCl₃:MeOH:H₂O=3:4:1.5); ¹H NMR(CD₃OD, 500 MHz) δ5.73 (d, J=7.3 Hz, 1 H), 4.72 (q, J=6.7 Hz, 1 H), 3.86(m, 1 H), 3.84 (d, J=11.6 Hz, 1 H), 3.74 (m, 1 H), 3.70 (m, 1 H), 3.66(dd, J=5.5, 11.6 Hz, 1 H), 3.45 (dd, J=9.8, 9.8 Hz, 1 H), 2.0 (s, 3 H),1.83 (d, J=7.3 Hz, 3 H); ¹³C NMR (CD₃OD, 500 MHz) δ180.4, 173.2, 93.7,77.7, 77.4, 74.2, 71.8, 62.0, 54.5, 22.2, 19.2; HRMS(FAB) calcd forC₁₁H₁₉NO₁₁P [M−H⁺]: 372.0696, found: 372.0711.

6.17.17 Compound 13b

To a solution of 2 (20 mg, 0.042 mmol) in 1 mL of CH₂Cl₂ is added DIEA(16 μL, 0.924 mmol). The reaction vessel is cooled to −30° C., thenMeOTf (5.2 μL, 0.046 mmol) is added. The reaction is complete afterstirring at room temperature for 30 min. Saturated NaHCO₃ is added. Themixture is extracted with CH₂Cl₂ (3×5 mL). The organic layers arecombined, dried over anhydrous sodium sulfate, filtered, concentratedand purified by flash chromatography (45% EtOAc/petroleum ether) to give18 mg (87%) of product as a white powder. The following chemistry is thesame as for 13a. R_(f)0.12 (CHCl₃:MeOH:H₂O=3:2:0.5); ¹H NMR (CD₃OD, 500MHz) δ5.50 (dd, J=3.4, 7.3 Hz, 1 H), 4.58 (q, J=6.7 Hz, 1 H), 3.87 (m, 2H), 3.84 (m, 1 H), 3.73 (3, 3 H), 3.62 (m, 2 H), 3.42 (dd, J=9.2, 9.2Hz, 1 H), 2.00 (s, 3 H), 1.37 (d, J=7.0 Hz, 3 H); ¹³C NMR (CD₃OD, 500MHz) δ176.4, 173.8, 95.0, 80.7, 77.3, 74.8, 72.8, 62.9, 54.9, 52.6,23.2, 19.4; ESI−MS calcd for C₁₂H₂₃NO₁₁P [M+H⁺]: 388,found: 388.

6.17.18 Compound 14

Compound 14a-c are made by the same approach as 1a, except by usingR—OPO₃PO(OPh)₂ instead of (R)-(+)-β-citronellol-OPO₃PO(OPh)₂. ESI-MS for14a C₃₂H₅₈N₇O₂₁P₂ [M+H⁺]: 938; ESI−MS for 14b C₃₃H₆₀N₇O₂₁P₂ [M+N⁺]: 952;ESI−MS for 14c C₃₄H₆₀N₇O₂₁P₂ [M+H⁺]: 964.

6.17.19 Compound 15

To a microfuge tube containing 1 equivalent 1b (10 μg) and 3 equivalents¹⁴C-UDP-GlcNAc in 100 μL HEPES reaction buffer (25 mM HEPES, pH 7.9, and2.5 mM MgCl₂) is added 1 μg purified MurG. The reaction is terminatedafter 30 minutes by heating MurG to 65° C. for five minutes. Thereaction is evaluated by transferring a 10 μL aliquot to a tubecontaining a 3-fold molar excess of Tetralink Tetrameric Avidin Resin(based on the amount of 1b expected in one tenth of a volume of thereaction mixture), diluting with H₂O, transferring the suspension to a96 well filter plate, and washing to remove unbound radioactivity asdescribed in more detail under the experimental for the initial rateassays. The resin is then transferred to a scintillation vial containingEcolite and counted. The conversion to disaccharide product 15 isestimated to be greater than 90% based on the counts incorporated intothe resin. The mixture containing 15 is suitable for evaluatingtransglycosylase activity.

¹H NMR assignments are made from 1D and 2D spectra (COSY)

¹H NMR (CD₃OD, 500 MHz) δ ppm 5.09 (t, J=5.0 Hz, 1 H, H-7), 3.90 (m, 2H, H-1), 1.99 (m, 2 H, H-6), 1.67 (m, 1 H, H-2), 1.65 (s, 3 H, H-9),1.62 (m, 1 H), H-3), 1.59 (s, 3 H, H-10), 1.41 (m, 1 H, H-2′), 1.34 (m,1 H, H-5), 1.16 (m, 1 H, H-5′), 0.91 (d, J=6.5 Hz, 3 H, H-4).

¹HNMR assignments are made from 1D and 2D spectra (COSY, ROESY)

¹H NMR (DMSO, 500 MHz) δ ppm 8.24 (d, J=7.5 Hz, 1 H, D-γ-Glu-NH), 8.18(d, J=8.5 Hz, 1 H, D-Ala₂-NH), 8.17 (d, J=7.0 Hz, 1 H, D-Ala₁-NH), 8.01(d, J=7.5 H 1 H, L-Lys-NH), 7.47 (d, J=7.5 Hz, 1 H, L-Ala-NH), 6.94 (t,J=5.0 Hz, 1 H, L-Lys-NHCOOR), 4.29 (m, 1 H, D-Ala₁-Hα), 4.24 (m, 1 H,D-Ala₂-Hα), 4.18 (m, 1 H, D-γ-Glu-Hα), 4.14 (m, 1 H, L-Lys-Hα), 4.12 (m,1 H, L-Ala-Hα), 3.58 (s, 3 H, D-Ala₂-COOCH ₃), 2.91 (m, 2 H, L-Lys-Hε),2.17 (m, 2 H, D-γ-Glu-Hγ), 1.92 (m, 1 H, D-γ-Glu-Hβ), 1.79 (m, 1 H,D-γ-Glu-Hβ′), 1.56 (m, 1 H, L-Lys-Hβ), 1.48 (m, 1 H, L-Lys-Hβ′), 1.35(m, 2 H, L-Lys-Hδ), 1.29 (d, J=7.0 Hz, 3 H, D-Ala₂-CH ₃), 1.23 (d, J=6.5Hz, 3 H, L-Ala-CH ₃), 1.22 (m, 1 H, L-Lys-Hγ), 1.19 (m, 1 H, L-Lys-Hγ′),1.19 (d, J=7.0 Hz, 3 H, D-Ala₁-CH ₃), 0.01-0.00 (s, 9 H; s, 9 H, TMS-CH₃).

¹H NMR assignments are made from 1D and 2D spectra (COSY, NOESY)

¹H NMR (DMSO, 500 MHz) δ ppm 8.36 (d, J=7.2 Hz, 1 H, L-Lys-NH), 8.21 (d,J=8.0 Hz, 1 H, D-Ala₂-NH), 8.19 (d, J=8.2 Hz, 1 H, D-Ala₁-NH), 8.10 (d,J=6.0 Hz, 1 H D-γ-Glu-NH), 7.32 (d, J=7.5 Hz, 1 H, L-Ala-NH), 6.95 (t,J=5.0 Hz, 1 H, L-Lys-NHCOOR), 5.26 (d, J=6.0 Hz, 1 H, H-1′), 5.07 (t,J=7.0 Hz, 1 H, H-7), 4.30 (m, 1 H, L-Ala-Hα), 4.27 (m, 1 H, D-Ala₂-Hα),4.23 (m, 1 H, D-Ala₁-Hα), 4.13 (m, 1 H, D-γ-Glu-Hα), 4.12 (m, 1 H,L-Lys-Hα), 4.12 (m, 1 H, H-7′), 3.87 (m, 1 H, H-2′), 3.77 (m, 2 H, H-1),3.62 (m, 1 H, H-5′), 3.60 (s, 3 H, D-Ala₂-COOCH ₃), 3.51 (m, 1 H, H-3′),3.33 (m, 1 H, H4′), 2.91 (m, 2 H, L-Lys-Hε), 2.17 (m, 2 H, D-γ-Glu-Hγ),1.94 (m, 2 H, H-6), 1.91 (m, 1 H, D-γ-Glu-Hβ), 1.51 (m, 1 H,D-γ-Glu-Hβ), 1.80 (s, 3 H, NHCOCH ₃-2′), 1.62 (s, 3 H, CH₃₋₉), 1.58 (s,3 H, CH₃-10), 1.50 (m, 1 H, H-3), 1.51 (m, 1 H, L-Lys-Hβ), 1.49 (m, 1 H,L-Lys-Hβ), 1.35 (m, 2 H, L-Lys-Hδ), 1.51 (m, 1 H, H-2), 1.27 (m, 1 H,H-2), 1.29 (d, J=7.2 Hz, 3 H, D-Ala₁-CH ₃), 1.19 (d, J=7.4 Hz, 3 H,D-Ala₂-CH ₃), 1.24 (d, J=5.5 Hz, 3 H, CH ₃-8′), 1.27 (m, 1 H, H-5), 1.11(m, 1 H, H-5), 1.25 (d, J=6.8 Hz, 3 H, L-Ala-CH ₃), 1.23 (m, 2 H,L-Lys-Hγ), 0.84 (d, J=6.5 Hz, 3 H, CH₃-4), 0.02-0.01 (s, 9 H; s, 9 H,TMS-CH ₃).

¹H NMR are made from 1D and 2D spectra (COSY).

¹H NMR (CD₃OD, 500 MHz), δ ppm 5.58 (1 H, H-1′), 5.11 (t, J=6.5 Hz, 1 H,H-7), 4.50-4.00 (L-Ala-Hα, D-γ-Glu-Hα, L-Lys-Hα, D-Ala_(1,2)-Hα, H-7′),4.10 (m, 1 H, H-2′), 3.98 (m, 1 H, H-5′), 3.87 (m, 1 H, H-6′), 3.80 (m,1 H, H-3′), 3.75 (m, 1 H, H-6′), 3.56 (m, 1 H, H-4′), 2.94 (m, 2 H,L-Lys-Hε), 2.34 (m, 2 H, D-γ-Glu-Hγ), 2.10 (s, 3 H, NHCOCH ₃-2′), 2.00(m, 1 H, D-γ-Glu-Hβ), 1.92 (m, 1 H, D-γ-Glu-Hβ), 1.98 (m, 2 H, H-6),1.74 (m, 2 H, L-Lys-Hδ), 1.67 (s, 3 H, CH₃-9), 1.62 (m, 1 H, H-3), 1.60(s, 3 H, CH₃-10), 1.50-1.39 (12 H. L-Ala-CH ₃, D-Ala_(1,2)-CH ₃, CH₃-7′), 1.23 (m, 2 H, L-Lys-Hγ), 0.93 (d, J=6.5 Hz, 3 H, CH₃-4).

¹HNMR are made from 1D and 2D spectra (COSY).

¹H NMR (CD₃OD, 500MHz) δ ppm 5.52 (d, J=4.5 Hz, 1 H, H-1′), 5.12 (t,J=7.0 Hz, 1 H, H-7), 4.50 (m, 1 H, H-b1), 4.39-4.19 (L-Ala-Hα,D-γ-Glu-Hα, L-Lys-Hα, D-Ala_(1,2)-Hα, H-7′), 4.31 (m, 1 H, H-b2), 4.20(m, 1 H, H-2′), 4.00 (m, 1 H, H-5′) 3.89 (m, 1 H, H-6′), 3.76 (m, 1 H,H-3′), 3.72 (m, 1 H, H-6′), 3.51 (m, 1 H, H4′), 3.22 (m, 1 H, H-b4),3.18 (m, 2 H, H-b9), 2.95 (dd, J=12.5, 5.0 Hz, 1 H, H-b3), 2.71 (d,J=12.5 Hz, 1 H, H-b3′), 2.27 (m, 2 H, D-γ-Glu-Hγ), 2.02 (s, 3 H, NHCOCH₃-2′), 2.01 (m, 2 H, H-6), 1.85 (m, 2 H, D-γ-Glu-Hβ), 1.67 (m, 2 H,H-b5), 1.67 (s, 3 H, CH ₃-9), 1.61 (s, 3 H, CH ₃-10), 1.62 (m, 1 H,H-3), 1.53 (m, 2 H, H-b10), 1.45-1.37 (12 H, L-Ala-CH ₃, D-Ala_(1,2)-CH₃, CH ₃-8′), 1.38 (m, 1 H, H-5), 1.17 (m, 1 H, H-5), 0.94 (d, J=6.8 Hz,3 H, CH ₃-4).

The preceding examples are provided as a further illustration of thepresent invention. The specific embodiments described above are not tobe construed to limit the invention in any way, which invention broadlyencompasses such embodiments, as well as those embodiments that would beevident to those of ordinary skill upon consideration of the disclosureherein provided. The invention is limited solely by the claims, whichfollow.

What is claimed is:
 1. A method of detecting GlcNAc transferase activityin a sample suspected of containing a protein or an active fragmentthereof exhibiting GlcNAc transferase activity comprising: contacting asample suspected of containing a protein or an active fragment thereofexhibiting GlcNAc transferase activity with effective amounts of labeledUDP-GlcNAc substrate and a substance comprising the chemical moiety ofthe formula:

 in which “R” is an acyl group comprising 2 or more carbon atoms, “R₁”is a substituted or unsubstituted alkyl group comprising 1 or morecarbon atoms, “R₂” is hydrogen or a substituted or unsubstituted alkylgroup comprising 1 or more carbon atoms, “A” is a substituted orunsubstituted amino acid residue or a peptide comprising 2 or moresubstituted or unsubstituted amino acid residues, “R₃” comprises asubstituted or unsubstituted alkyl group comprising 10 to 40 carbonatoms, pyrophosphate protecting groups and pharmaceutically acceptablesalts thereof, provided that said substance is not Lipid I, the naturalsubstrate of wild type MurG enzyme, under conditions effective toprovide a labeled coupling product comprising labeled GlcNAc coupled tosaid substance via a glycosidic bond in the presence of a protein or anactive fragment thereof exhibiting GlcNAc transferase activity;detecting the formation or presence of said labeled coupling product,which is indicative of GlcNAc transferase activity in said sample. 2.The method of claim 1 in which said labeled GlcNAc substrate is labeledUDP-GlcNAc.
 3. The method of claim 2 in which at least a portion of saidsample comprises a portion of a lysed bacterial culture, a portion of asupernatant thereof, a portion of a membrane fraction thereof, a portionof a protein fraction thereof, a purified enzyme, a soluble enzyme,purified or synthesized lipid or mixtures of same.
 4. The method ofclaim 1 in which the detection step comprises separation of labeledcoupling product from labeled UDP-GlcNAc substrate.
 5. The method ofclaim 1 in which said detection step comprises binding said “A” or “R₃”to a solid support via a biotin tag, wherein said solid support includesan avidin or streptavidin coated resin.
 6. The method of claim 5 whereinsaid detection step provide a continuous monitoring of product formationvia the use of scintillation proximity assay.